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.

Glucocorticoids paradoxically promote steroid resistance in B cell acute lymphoblastic leukemia through CXCR4/PLC signaling.

Glucocorticoid (GC) resistance in childhood relapsed B-cell acute lymphoblastic leukemia (B-ALL) represents an important challenge. Despite decades of clinical use, the mechanisms underlying resistance remain poorly understood. Here, we report that in B-ALL, GC paradoxically induce their own resistance by activating a phospholipase C (PLC)-mediated cell survival pathway through the chemokine receptor, CXCR4. We identify PLC as aberrantly activated in GC-resistant B-ALL and its inhibition is able to induce cell death by compromising several transcriptional programs. Mechanistically, dexamethasone (Dex) provokes CXCR4 signaling, resulting in the activation of PLC-dependent Ca2+ and protein kinase C signaling pathways, which curtail anticancer activity. Treatment with a CXCR4 antagonist or a PLC inhibitor improves survival of Dex-treated NSG mice in vivo. CXCR4/PLC axis inhibition significantly reverses Dex resistance in B-ALL cell lines (in vitro and in vivo) and cells from Dex resistant ALL patients. Our study identifies how activation of the PLC signalosome in B-ALL by Dex limits the upfront efficacy of this chemotherapeutic agent.

Deep learning assists in acute leukemia detection and cell classification via flow cytometry using the acute leukemia orientation tube.

This study aimed to evaluate the sensitivity of AI in screening acute leukemia and its capability to classify either physiological or pathological cells. Utilizing an acute leukemia orientation tube (ALOT), one of the protocols of Euroflow, flow cytometry efficiently identifies various forms of acute leukemia. However, the analysis of flow cytometry can be time-consuming work. This retrospective study included 241 patients who underwent flow cytometry examination using ALOT between 2017 and 2022. The collected flow cytometry data were used to train an artificial intelligence using deep learning. The trained AI demonstrated a 94.6% sensitivity in detecting acute myeloid leukemia (AML) patients and a 98.2% sensitivity for B-lymphoblastic leukemia (B-ALL) patients. The sensitivities of physiological cells were at least 80%, with variable performance for pathological cells. In conclusion, the AI, trained with ResNet-50 and EverFlow, shows promising results in identifying patients with AML and B-ALL, as well as classifying physiological cells.

Aggressive precursor B cell ALL of cervix with obstructive uropathy.

Involvement of the cervix with acute lymphoblastic leukaemia (ALL) is extremely rare. In this case report, we discuss an unmarried woman in her early 20s, who presented in the emergency with lower abdominal pain and irregular vaginal bleeding for 1 month. Clinical examination and imaging revealed a large cervical mass probably neoplastic with obstructive uropathy. On evaluation, she was diagnosed incidentally with CALLA-positive precursor B cell ALL in peripheral blood flow cytometry. Involvement of B cell ALL in cervical mass was confirmed by histopathological examination of cervical biopsy and immunohistochemistry markers. Her history was not suggestive of signs and symptoms pertaining to leukaemia. Literature is sparse with only a few cases reporting cervical leukaemic infiltration. The present case report is a rarest case where the primary/initial presentation of precursor B cell ALL was seen with cervical involvement and obstructive uropathy mimicking characteristics of advanced cervical malignancy.

The Eµ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia.

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Systemic xanthogranuloma involving bone marrow and skin in a case of B-Lymphoblastic Leukemia.

Juvenile xanthogranuloma is a benign self-limiting lesion commonly described in infants and young children. It most commonly involves the skin presenting as single or multiple yellowish-brown papules. Clinical scenario with the classic histomorphology showing histiocytic aggregates in the dermis with xanthomatous cytoplasm, toutan type giant cells, immunohistochemistry with positive CD68, CD163, factor XIIIa and negative CD1a and S-100 help in diagnosis. However, diagnosis becomes challenging with predominant systemic bone marrow involvement in post-B-lymphoblastic leukemia settings.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Clinicopathologic features of relapsed CD19(-) B-ALL in CD19-targeted immunotherapy: Biological insights into relapse and LILRB1 as a novel B-cell marker for CD19(-) B lymphoblasts.

INTRODUCTION: The mechanism of relapsed CD19(-) B-ALL after anti-CD19 immunotherapy (Kymriah [CART-19] and blinatumomab) is under active investigation. Our study aims to assess LILRB1 as a novel B-cell marker for detecting CD19(-) B-lymphoblasts and to analyze the clinicopathologic/genetic features of such disease to provide biological insight into relapse. METHODS: Six patients (3 males/3 females, median age of 14 years) with relapsed CD19(-) B-ALL were analyzed for cytogenetic/genetic profile and immunophenotype. RESULTS: CD19(-) B-ALL emerged after an interval of 5.8 months following anti-CD19 therapy. Five of six patients had B-cell aplasia, indicative of a persistent effect of CART or blinatumomab at relapse. Importantly, LILRB1 was variably expressed on CD19(-) and CD19(+) B lymphoblasts, strong on CD34(+) lymphoblasts and dim/partial on CD34(-) lymphoblasts. Three of six patients with paired B-ALL samples (pre- and post-anti-CD19 therapy) carried complex and different cytogenetic abnormalities, either as completely different or sharing a subset of cytogenetic abnormalities. CONCLUSION: LILRB1 can be used as a novel B-cell marker to identify CD19(-) B lymphoblasts. The emergence of CD19(-) B-ALL appears to be associated with complex cytogenetic evolutions. The mechanism of CD19(-) B-ALL relapse under anti-CD19 immune pressure remains to be explored by comprehensive molecular studies.

Blinatumomab in Practice.

PURPOSE OF REVIEW: Acute lymphoblastic leukemia (ALL) is a rare hematologic neoplasm in adults, with most cases defined by pathology related to abnormal B cell proliferation known as B-cell ALL. The course is challenging, with less-than-optimal survival outcomes, even with aggressive multiagent chemotherapy and consideration for stem cell transplantation. Novel therapies focused on targetable pathways are being investigated to improve outcomes while simultaneously decreasing toxicity. In our review, we aim to evaluate the utilization of blinatumomab in B-cell ALL and provide insight on how this guides our management. RECENT FINDINGS: Blinatumomab is a bispecific T-cell engager (BiTE) immunotherapy that neutralizes malignant cells by instigating CD3-positive T cells to target CD19-positive B cells. However, this therapy targets both malignant and non-malignant lymphocytes with potentially severe side effects such as cytokine release syndrome or neurotoxicity. Evidence evaluating utilization in the relapsed or refractory setting has been most supported; however, newer trials have also indicated improved survival in the frontline treatment of B-cell ALL. As this therapy is relatively new, the treatment team may include members who are less experienced with the typical treatment course and drug mechanics. This review synthesized available data investigating the effectiveness of blinatumomab effectiveness and its adverse events in addition to providing guidance on safe administration methods utilizing a multidisciplinary healthcare team. When care is coordinated in these settings, serious side effects can be recognized early, allowing for necessary intervention leading to improved quality of life and overall survival. Future research will continue to evaluate blinatumomab in different lines of therapy and expand its way into community settings.

INSPIRED Symposium Part 4A: Access to CAR T Cell Therapy in Unique Populations with B Cell Acute Lymphoblastic Leukemia.

The approval of tisagenlecleucel (tisa-cel) for use in children with B cell acute lymphoblastic leukemia (B-ALL) was based on the phase 2 ELIANA trial, a global registration study. However, the ELIANA trial excluded specific subsets of patients facing unique challenges and did not include a sufficient number of patients to adequately evaluate outcomes in rare subpopulations. Since the commercialization of tisa-cel, data have become available that support therapeutic indications beyond the specific cohorts previously eligible for chimeric antigen receptor (CAR) T cells targeted to CD19 (CD19 CAR-T) therapy on the registration clinical trial. Substantial real-world data and aggregate clinical trial data have addressed gaps in our understanding of response rates, longer-term efficacy, and toxicities associated with CD19 CAR-T in special populations and rare clinical scenarios. These include patients with central nervous system relapsed disease, who were excluded from ELIANA and other early CAR-T trials owing to concerns about risk of neurotoxicity that have not been born out. There is also interest in the use of CD19 CAR-T for very-high-risk patients earlier in the course of therapy, such as patients with persistent minimal residual disease after 2 cycles of upfront chemotherapy and patients with first relapse of B-ALL. However, these indications are not specified on the label for tisa-cel and historically were not included in eligibility criteria for most clinical trials; data addressing these populations are needed. Populations at high risk of relapse, including patients with high-risk cytogenetic lesions, infants with B-ALL, patients with trisomy 21, and young adults with B-ALL, also may benefit from earlier treatment with CD19 CAR-T. It is important to prospectively study patient-reported outcomes given the differential toxicity expected between CD19 CAR-T and the historic standard of care, hematopoietic cell transplantation. Now that CD19 CAR-T therapy is commercially available, studies evaluating potential access disparities created by this very expensive novel therapy are increasingly pressing.

Increased Incidence of TdT-negative Pre-B Acute Lymphoblastic Leukemia Associated With Poor Prognostic Features Among Mexican Children in Central Mexico.

Mexican and Hispanic children in Mexico and the United States, respectively, have the highest incidence and worst outcomes of pre-B acute lymphoblastic leukemia (ALL) compared with other racial/ethnic groups. Terminal deoxynucleotidyl transferase (TdT) is an intranuclear DNA polymerase normally present on immature lymphocytes (TdT-positive) and distinguishes ALL from mature lymphoid malignancies. We performed a multisite retrospective study to determine the incidence of TdT-negative precursor B-cell acute lymphoblastic leukemia (pre-B ALL) among Mexican, Caucasian, and US-born Hispanic children to correlate TdT expression with patient characteristics and known prognostic factors. Fisher exact test was performed for categorical variables and the Wilcoxon rank-sum test was used for continuous variables. TdT-negative pre-B ALL was most frequently identified in patients with National Cancer Institute high-risk disease ( P =0.014). TdT-negative expression was also most frequently associated with hypodiploid pre-B ALL ( P =0.001) and KMT2A gene rearrangement ( P =0.0012). Mexican children had the highest incidence of TdT-negative ALL compared with Caucasians and US Hispanics ( P <0.001), with an increased incidence of poor prognostic features as well. This study demonstrates significant differences in TdT-negative expression, genomic alterations, and leukemic ploidy based on race and ethnicity.

Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish.

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression.

Preclinical development of 1B7/CD3, a novel anti-TSLPR bispecific antibody that targets CRLF2-rearranged Ph-like B-ALL.

Patients harboring CRLF2-rearranged B-lineage acute lymphocytic leukemia (B-ALL) face a 5-year survival rate as low as 20%. While significant gains have been made to position targeted therapies for B-ALL treatment, continued efforts are needed to develop therapeutic options with improved duration of response. Here, first we have demonstrated that patients with CRLF2-rearranged Ph-like ALL harbor elevated thymic stromal lymphopoietin receptor (TSLPR) expression, which is comparable with CD19. Then we present and evaluate the anti-tumor characteristics of 1B7/CD3, a novel CD3-redirecting bispecific antibody (BsAb) that co-targets TSLPR. In vitro, 1B7/CD3 exhibits optimal binding to both human and cynomolgus CD3 and TSLPR. Further, 1B7/CD3 was shown to induce potent T cell activation and tumor lytic activity in both cell lines and primary B-ALL patient samples. Using humanized cell- or patient-derived xenograft models, 1B7/CD3 treatment was shown to trigger dose-dependent tumor remission or growth inhibition across donors as well as induce T cell activation and expansion. Pharmacokinetic studies in murine models revealed 1B7/CD3 to exhibit a prolonged half-life. Finally, toxicology studies using cynomolgus monkeys found that the maximum tolerated dose of 1B7/CD3 was ≤1 mg/kg. Overall, our preclinical data provide the framework for the clinical evaluation of 1B7/CD3 in patients with CRLF2-rearranged B-ALL.