Acute Lymphoblastic Leukemia Patients Research Articles

Anti-T-lymphocyte globulin exposure is associated with acute graft-versus-host disease and relapse in pediatric acute lymphoblastic leukemia patients undergoing hematopoietic stem cell transplantation: a multinational prospective study.

Anti T-lymphocyte globulin (ATLG) is used in hematopoietic stem cell transplantation (HSCT) to prevent graft-versus-host disease (GvHD) and graft failure. To date, insight in ATLG pharmacokinetics and -dynamics (PK/PD) is limited, and population PK (POPPK) models are lacking. In this prospective study, we describe ATLG POPPK using NONMEM® and the impact of ATLG exposure on clinical outcome and immune reconstitution in a homogeneous cohort of pediatric acute lymphoblastic leukemia (ALL) patients transplanted with a matched unrelated donor and receiving uniform ATLG dosing. Based on 121 patients and 812 samples for POPPK analysis, a two-compartmental model with parallel linear and non-linear clearance and bodyweight as covariate, best described the ATLG concentrationtime data. The level of ATLG exposure (day active ATLG day 16 8.2%, p.

Enrichment of T-Lymphocytes from Leukemic Blood using Inertial Microfluidics towards Improved Chimeric Antigen Receptor-T Cell Manufacturing

Chimeric antigen receptor (CAR-T) cell therapy is a successful immunotherapy for treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia (CLL) patient's samples. In healthy donors used to optimise the process, the lymphocyte purity was enriched from 65% (SD±0.2) to 91% (SD±0.06) and T-cell purity was enriched from 45% (SD±0.1) to 73% (SD±0.02). Leukemic samples had higher starting B-cell compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells was achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD±0.09), 89% (SD±0.1) and 74% (SD±0.09) respectively, and a 70% (SD±0.1) T-cell recovery. CLL samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the CAR-T manufacturing workflow.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Rituximab potentially improves clinical outcomes of CAR-T therapy for r/r B-ALL via sensitizing leukemia cells to CAR-T-mediated cytotoxicity and reducing CAR-T exhaustion.

Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy. We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375mg/m2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry. The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p=0.0342; 41.7% vs. 25%, p=0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19-22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab-treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group. Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion.

Linkage of cell division cycle 42 with clinical features, treatment response and survival in adult Philadelphia chromosome negative acute lymphoblastic leukemia

Cell division cycle 42 (CDC42) regulates the progression of leukemia via mediating proliferation and immune evasion of malignant cells. The study aimed to investigate the correlation of CDC42 with clinical features, treatment response, event-free survival (EFS) and overall survival (OS) in adult Philadelphia chromosome negative acute lymphoblastic leukemia (Ph− ALL) patients. CDC42 expression in bone marrow mononuclear cells was detected in 78 adult Ph− ALL patients and 10 donors using real-time reverse transcriptase-polymerase chain reaction. CDC42 was increased in adult Ph− ALL patients compared with donors (p < .001). Besides, elevated CDC42 was linked with pro-B ALL or early-T ALL (p = .038) and white blood cell (WBC) elevation at diagnosis (p = .025). Fifty (64.1%) and 23 (29.5%) patients had complete remission (CR) at 1 month and minimal residual disease (MRD) after CR, respectively. CDC42 was inversely associated with CR at 1 month (p = .034), but not MRD after CR (p = .066). Concerning survival, patients with CDC42 ≥ 3.310 (cut by median value in patients) showed a shortened EFS (p = .006) and OS (p = .036) compared to those with CDC42 < 3.310. In detail, patients with CDC42 ≥ 3.310 and CDC42 < 3.310 had 5-year EFS rate of 29.9% and 45.4%, and 5-year OS rate of 39.4% and 63.6%, correspondingly. Further multivariate Cox’s regression analyses revealed that CDC42 ≥ 3.310 was independently related to shorter EFS (hazard ratio = 2.933, p = .005). Elevated CDC42 is related with pro-B ALL or early-T ALL, WBC elevation at diagnosis, unfavorable treatment response and worse survival in adult Ph− ALL patients.

A rare KMT2A::CBL transcript in an acute monoblastic leukemia patient with an unfavorable outcome

BackgroundLysine [K] methyltransferase 2A (KMT2A, previously known as MLL) gene rearrangements are common in acute leukemias of various lineages and are associated with features such as chemotherapy resistance and rapid relapse. KMT2A::CBL is a rare fusion of unknown pathogenesis generated by a unique interstitial deletion of chromosome 11 that has been reported across a wide age range in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. The leukemogenic effect of the KMT2A::CBL rearrangement and its association with clinical prognosis have not been well clarified.Methods and resultsWe report the case of a 64-year-old female who was diagnosed with acute monoblastic leukemia (M5a) and who acquired the rare KMT2A::CBL fusion. The patient received multiple cycles of therapy but did not achieve remission and eventually succumbed to severe infection and disease progression. Additionally, we characterized the predicted KMT2A-CBL protein structure in this case to reveal the underlying leukemogenic mechanisms and summarized reported cases of hematological malignancies with KMT2A::CBL fusion to investigate the correlation of gene rearrangements with clinical outcomes.ConclusionsThis report provides novel insights into the leukemogenic potential of the KMT2A::CBL rearrangement and the correlation between gene rearrangements and clinical outcomes.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Evaluation of FOXP3 and IL10 as immunosuppressant markers in pediatric acute lymphocytic leukemia patients in Iraq

Background. Increased FOXP3+ Treg levels in the TME were positively correlated with a worse prognosis in certain cancer patients. FOXP3 was significantly overexpressed in pediatric B-ALL patients, and this overexpression was associated with a worse prognosis and increased risk of disease relapse. It is unknown if children that have the condition would have different prenatal immune development. This study investigates the relationship between IL10, and immunological development in pediatric ALL. Methods. The 70 blood samples from children between the ages of 2 and 14 for both sexes were obtained from patients with Acute lymphoblastic leukemia. Furthermore, this study comprised 54 healthy controls. Included in this study, FOXP3 expression on leukemic blast cells were assessed using flow cytometry. And IL10 were assessed using ELISA test. Results. Increase of FOXP3 cells was noticed in children with ALL compared to healthy individuals with significant increase of FOXP3 (mean ± SD, 91.156 ± 12.255 vs. 14.88 ± 7.897pg/ml (p &lt;0.05). In light of these findings, significant differences in the levels of FOXP3, which was higher in induction than consolidation stage of chemotherapy (mean ± SD, 91.24± 11.078vs. 86.35±5. 16.66) pg/ml (p &lt;0.05). Additionally, it was found that the prevalence of lymphoblast that express FOXP3 was notably higher in the HR group in contrast to the SR group (mean ± SD, 95.52± 5.79 vs. 42.88± 31.38) pg/ml (p &lt;0.05). Following these findings, significant differences in the levels of FOXP3 was higher in relapse than new diagnosis chemotherapy stage (mean ± SD, 95.66± 5.167 vs. 91.035±5. 13.177) pg/ml (p&lt;0.05). On the other hand, children with ALL were also found to have significantly higher levels of IL10 in the current study when compared to healthy controls (mean ± SD, 0.146 ± 0.145 vs. 0.076 ± 0.10 pg/ml (P &lt;0.05). Following these findings, significant differences in the levels of IL10 was higher in consolidation than induction chemotherapy stage (mean ± SD, 0.165± 0.084 vs.0 .0720±.018) pg/ml (p &lt;0.05), the serum levels of IL-10 were evaluated in the two groups: new diagnosis and relapse cases. As in the description profile, serum levels are higher in the relapse group with a highly significant difference. For the parameter IL10 (P ≤0.05). As for he mean level, it was as follows: of IL10 (0.130) pg/ml and (0.216) pg/ml, with (P-value =0.0001) in new diagnosis and relapse receptivity. Conclusions. According to our findings, B-ALL patients have significant amounts of both FOXP3 and IL10. This pilot study provides a novel way to look into the process mediating the appearance of these markers in a greater number of B-ALL patients at various stages of their treatment.

Outcomes and adverse events in older acute lymphoblastic Leukemia patients treated with a pediatric-inspired protocol with Pegylated or native Asparaginase

ABSTRACT This retrospective report presents the outcomes and adverse events (AEs) observed in 73 patients aged 60 years or older diagnosed with Philadelphia Chromosome-negative Acute Lymphoblastic Leukemia (Ph-negative ALL) treated with a pediatric-inspired protocol incorporating either Pegylated (PEG-ASP) or Native Asparaginase (EC-ASP). Notably, 61% of patients experienced AEs of Grade III-IV severity. The most prevalent AEs included thrombosis (35.6%), febrile neutropenia (38.4%), and transaminitis (34.2%). AEs did not translate into significant differences concerning overall survival, leukemia-free survival, or early mortality. Furthermore, we observed a reduction in early mortality rates (11% vs. 20%) and an increase in median overall survival (54 vs. 48 months) compared to our previous data. These findings suggest that the utilization of a pediatric-inspired chemotherapy protocol, with ASP, is an effective and well-tolerated therapeutic option for older patients with Ph-negative ALL. However, it emphasizes the importance of diligent monitoring and close follow-up throughout treatment.

Abstract 259: Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) patients who experience relapse have a dismal prognosis, with 5-year overall survival rates of 10% for adults and 36% for children. Relapse is attributed to cytotoxic chemotherapy’s inability to target Leukemia Initiating Cells (LICs) that have the potential to re-populate the cancer. There is a need for novel therapeutic strategies to target LIC self-renewal in T-ALL with improved safety profiles and better chances of transition to clinical development. This project utilized a transgenic zebrafish model that accurately recapitulates the most aggressive form of the human T-ALL. This model offered several advantages, including a high frequency of LICs and suitability for drug testing. Over 770 FDA-approved drugs were screened, in vivo using &amp;gt;2,500 syngeneic CG1 zebrafish. Further secondary screening in human T-ALL cells narrowed down the potential hits into 4 compounds that specifically interfere with self-renewal. Finally, the hit drug Amiloride was confirmed by a limiting dilation assay (LDA), resulting in a more than 4-fold decrease in the LIC frequency (p=0.0026). The top hit, Amiloride, which reduced the frequency of LICs in zebrafish and human T-ALL cells, is an inhibitor of the Sodium Hydrogen Exachanger-1 (NHE1). NHE1 has been previously linked to the proliferation, migration, and drug resistance of different cancers. In addition, Amiloride treatment was found to impair mitochondrial function in cancer cells, offering a possible mechanism for the inhibition of LICs. Research has not yet investigated how NHE1, and the altered mitochondrial dynamics related to its inhibition affect the development and persistence of LICs in T-ALL. In our human T-ALL cells, we found that Amiloride significantly reduced mitochondrial basal respiration (p=0.0074), ATP production (p=0.0008) and increased the expression of genes associated with mitochondrial stress such as TFAM, PINK1, PARKIN and MT-CO1. Experiments using shRNA mediated knock down of the NHE1 in human T-ALL cells are underway and will be followed by investigating the impact of NHE1 inhibition on primary patient-derived T-ALL cells in a mouse model. Overall, this project showcases of the power of the zebrafish in cancer research in an efficient and accurate large-scale screen for drugs that impact LIC self-renewal. We have identified Amiloride as a top hit that reduces the frequency of LIC in vivo and in vitro. Finally, we are continuing to investigate the role of the NHE1 in self-renewal in human T-ALL and the associated mitochondrial alterations. Citation Format: Majd Al-Hamaly, Yelena Chernyavskaya, Emma Arvin, Logan Turner, Meghan Haney, Jessica Blackburn. Zebrafish empowered large-scale drug screen to uncover novel targets of self-renewal in T-cell acute lymphoblastic leukemia initiating cells [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 259.

Abstract 5956: FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients

Abstract Backgroud: The prognosis of B cell ALL patients (pts) relapsing after Hematopoietic Transplant is poor and new drugs are needed. In acute myeloid leukemia, FLT3-inhibitors (FLT3i) are showing promising results in FLT3-mutated (mut) cases. Their potential use in FLT3-mut B-ALL has been poorly investigated. Aim: To assess if FLT3 could be a valid targetable marker in B-ALL. Methods: We sequenced with a capture based large RNA-seq panel (1385genes) 183 adult B-ALL [n=52 Pos for Ph or t(4;11) or t(1;19) and n=131 Triple-Negative (TN) pts that are negative for Ph (n=43), t(4;11) and t(1;19) translocations] and 15 donors. To confirm FLT3 muts, 13 available samples were further sequenced with Extended Myeloid Solution (98 genes; SOPHiA) where FLT3 gene is fully covered. In vitro studies using 5 FLT3i [Gilteritinib (Gil), Midostaurin, Crenolanib, Sorafenib and Quizartibin] and Venetoclax (Ven) were conducted on pt primary cells and on TN and other B-ALL cell lines (MUTZ5, MHH-CALL4 Ph-like; NALM6;RS4;11, 697, REH) and 2 AML cell lines (OCI-AML3 FLT3;MV-4-11 FLT3-ITD). Results: We found 15 FLT3 mut in 14/131 TN B-ALL and in 1/43 Ph+ cases: 43.8% were TKD mut, 3 ITD mut, 3 splicing site mut,1 N-terminal, 1 juxta-membrane domain and 1 Ig-like site (Fig. 1A). After pathogenicity interpretation we excluded 3 variants and one mutation was not confirmed at DNA level. Overall 7.6% of TN were mut (Ph+ 2.3%). 81.8% of FLT3mut pts potentially druggable with FLT3i, mainly TKD (63.3%). Targeted NGS revealed the co-occurrence with other mutations (range 2-15), and in particular with CSMD1 (n=4), KDM6A, KMT2D and CREBBP (n=3) alterations. Moreover, 11/13 samples carried copy number alteration (mean 12), mostly amplifications. CDKN2A &amp; IKZF1 were the most frequently altered genes (n=5;4 CN-loss). FLT3 expression was increased in 11/15 cases compared to donors and wt pts. As reported, the expression was higher in ZNF384 or MLLr B-ALL subtypes.To evaluate the effect of FLT3is in ALL, we treated ex vivo primary leukemic cells of 6 adult ALL patients (FLT3-mut n=4; FLT3-wt n=2) with increasing concentrations of 5 FLT3i for 24, 48 and 72h. We observed a trend towards greater response of FLT3-mut ALL cells compared to wt ones. The average IC50 values in response to the five FLT3i was 1.6 uM and 24.5 uM after 72h of treatment in FLT3-mut and FLT3-wt B-ALL, respectively (Fig. 1B). We than evaluated the sensitivity to the 5 Inhs in all cell lines. Interestingly MHH-CALL4 wt were more sensitive to Gil compared to OCI AML3 wt, and showed the same effect compare to MV-4-11 AML mut (F.1C). Given the effect of Gil in single agent, we combined it with Ven in wt B-ALL cells at increasing concentrations. Interestingly We noticed a strong additive effect with the higher Gil concentration and all Ven doses(F.1D). Conclusions: FLT3 alterations identify a novel subgroup of TN B-ALL with therapeutic potential also in combination regimens. Ricerca Corrente Italian MoH L3P1946. Citation Format: Anna Ferrari, Andrea Ghelli Luserna di Rorà, Federico Lo Schiavo, Eugenio Fonzi, Giorgia Capirossi, Chiara Salvesi, Maria Teresa Bochicchio, Lorenzo Ledda, Chiara Servilli, Matteo Paganelli, Giovanni Marconi, Cristina Papayannidis, Chiara Sartor, Michela Rondoni, Anna Maria Mianulli, Barbara Giannini, Crescenza Pasciolla, Fabio Giglio, Sara Galimberti, Monica Fumagalli, Prassede Salutari, Valentina Gaidano, Giovanni Martinelli, Giorgia Simonetti. FLT3 alterations and inhibition in triple negative B-cell adult acute lymphoblastic leukemia patients [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 5956.

Abstract 6672: Unraveling the microbiome: antibiotic impact on CAR T therapy and the synergistic potential of vancomycin in enhancing antitumor immunity

Abstract The landscape of treating hematologic malignancies has undergone a revolutionary transformation with the advent of Anti-CD19 chimeric antigen receptor T cell therapy (CART-19). Despite its success, persistent challenges like disease relapse and CAR-mediated toxicities remain. A comprehensive multicenter study involving 228 individuals revealed a significant association between antibiotic administration, particularly piperacillin/tazobactam, meropenem, and imipenem/cilastatin (P-I-M), targeting anaerobes within four weeks preceding CART-19 therapy, and adverse outcomes, including worsened survival and increased neurotoxicity. Intriguingly, another antibiotic, Cefepime, also used for neutropenic fever, displayed a positive trend when compared with P-I-M. This highlights the intricate relationship between the modulation of the intestinal microbiome and outcomes in CART-19 therapy for B cell malignancies. Building on these critical observations, our study delved into the impact of vancomycin-induced gut microbiota dysbiosis on chimeric antigen receptor (CAR) T immunotherapy. Utilizing preclinical models of CD19+ A20 lymphoma and CD19+ B16 melanoma, we discovered that mice receiving vancomycin alongside CART-19 therapy exhibited enhanced tumor control and increased cross-presentation of tumor-associated antigens (TAAs) compared to those receiving CART-19 alone. This effect was further validated through fecal microbiota transplants from healthy human donors to pre-conditioned mice, replicating the favorable findings. Importantly, our study extends to clinical correlates, as B cell acute lymphoblastic leukemia patients treated with CART-19 and oral vancomycin displayed elevated CART-19 peak expansion compared to unexposed patients. These compelling results underscore the profound influence of the gut microbiota on CAR T cell therapy and suggest that vancomycin-induced gut microbiota modulation may emerge as a potent strategy to enhance therapeutic outcomes across various tumor types. Citation Format: Andrea Facciabene, Mireia Uribe-Herranz, Vijay Bhoj. Unraveling the microbiome: antibiotic impact on CAR T therapy and the synergistic potential of vancomycin in enhancing antitumor immunity [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 6672.

Abstract 6703: Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies

Abstract The advent of T Cell Engager (TCE) immunotherapies marks a transformative shift in cancer treatment paradigms, introducing a novel action mechanism. Blinatumomab, a bispecific CD19/CD3 TCE, has shown remarkable efficacy in treating refractory or relapsed B-cell Acute Lymphoblastic Leukaemia (B-ALL), including cases resistant to conventional chemotherapy, leading to its global approval for B-ALL management. Despite its pronounced clinical success, Blinatumomab faces challenges with patient adherence, primarily due to the protein’s short half-life. This necessitates continuous administration requested 24 hours injection throughout the treatment course. Additionally, the drug’s association with the induction of cytokine release syndrome (CRS), presumably linked to the hyperactivation of the cytokine network, presents a significant concern. To address these limitations, we leverage mRNA modality to develop innovative therapeutic mRNAs encoding CD19/CD3 TCE. These mRNA drugs hold the potential to improve protein exposure in vivo with a steady production of encoded protein in-situ. On the other hand, with the gradual expression of protein, mRNA shifted the transient, “pulse”-like pharmacokinetic (PK) feature of the protein drugs to a more “plateau”PK curve, which allows a natural priming of T cells to reduce the risk of CRS induction. In this work, we developed a series of mRNAs encoding CD19/CD3 TCE through our mRNA technology platform and LNP delivery platform. Comparative analyses demonstrated that the TCEs produced in vivo by these mRNAs match the structure and function of recombinantly expressed proteins. PK studies in both mice and non-human primates confirmed a favorable PK profile with over 10-fold increase in TCE exposure compared to their protein counterparts. This increased exposure correlated with superior antitumor efficacy in both ALL and lymphoma models, evidenced by the potent effects of single mRNA doses compared to multiple doses of protein-based treatments. Meanwhile IL6 cytokines induced in mRNA treated mice were significantly alleviated than those in protein treated animals, indicating a reduced risk of induction of CRS. One candidate mRNA demonstrated an optimal "plateau" pharmacokinetic profile, achieving extensive target cell eradication over 20 days with a single low dose of 0.001 mpk in non-human primates. An investigator-initiated clinical study with mRNA encoding CD19/CD3 TCE is underway in relapsed and/or refractory acute lymphoblastic leukemia patients. The preliminary data from the on-going study showed ABO2015 with an acceptable safety profile, and activation of peripheral T cells was observed in one patient with low dose. These findings underscore the significant clinical potential of mRNA-encoded CD19/CD3 TCE in treating B-cell malignancies, heralding a paradigm shift in the clinical application of protein-based T cell engagers. Citation Format: Xia Zhong, Liang Du, Zhenxing Yang, Xiaoli Xu, Jijun Yuan, Bo Ying. Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies [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 6703.

The influence of WeChat education and care program on anxiety, depression, insomnia, and general state of health in parents of pediatric acute lymphoblastic leukemia patients

PurposeWeChat-based education and care program serves as a promising nursing method for relieving mental stress in parents of pediatric patients. This study purposed to explore the influence of the WeChat education and care program (WECP) on mental health, insomnia, and general state of health in parents of pediatric acute lymphoblastic leukemia (ALL) patients.MethodsTotally, 146 parents of 73 primary pediatric ALL patients were randomized into the WECP group (74 parents of 37 patients) and standard care (SC) group (72 parents of 36 patients) to receive a 6-month corresponding intervention. Self-rating anxiety scale (SAS), self-rating depression scale (SDS), Athens insomnia scale (AIS), and 12-item general health questionnaire (GHQ-12) were assessed in parents of patients.ResultsSAS scores at the third month (M3) (P = 0.041) and M6 (P = 0.032) were reduced in WECP group versus SC group. SAS-defined anxiety rate at M6 (P = 0.035) was declined in WECP group versus SC group. SDS score at M6 was descended in WECP group versus SC group (P = 0.024). However, there was no discrepancy in SDS-defined depression rate at any time point between groups (all P > 0.05). AIS scores at M1 (P = 0.015) and M6 (P = 0.021), as well as GHQ-12 scores at M3 (P = 0.007) and M6 (P = 0.001) were decreased in WECP group versus SC group. By subgroup analyses, WECP exhibited good effects at M6 in mothers, but not in fathers.ConclusionWECP is a feasible and efficacy intervention to improve mental stress and health status among parents of pediatric ALL patients, especially in mothers.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rβγ stimulation, whereas the L45E mutation optimized IL-15Rβγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Relevance of CD20 antigen expression among paediatric patients with B-lineage acute lymphoblastic leukaemia.

Literature regarding prognostic relevance of CD20 antigen expression among paediatric B-lineage acute lymphoblastic leukaemia (B-ALL) patients is sparse and contradictory. We analysed clinical laboratory parameters and survival characteristics pertinent to CD20 expression among 224 treatment-naïve paediatric B-ALL patients. 50% patients had CD20 expression (CD20+ B-ALL). There was no difference in the clinical & laboratory presentation and end of induction measurable residual disease (EOI-MRD) status according to CD20 expression. As compared to CD20- B-ALL patients, CD20+ B-ALL patients had two times more relapse (16% vs. 29%, p = 0.034), inferior relapse-free survival (79% vs. 66%, p = 0.025) but no difference in overall survival (75% vs. 69%, p = 0.126). Similar to high-risk NCI status and EOI-MRD positivity, CD20 expression was an independent predictor for inferior relapse-free survival (HR: 1.860, 95% CI: 1.008-3.432, p = 0.047). Compared to baseline, there was a significant increase in CD20-expressing EOI-residual blasts among CD20- B-ALL patients (5% vs. 13%, p = 0.001). EOI residual blasts of both CD20+ and CD20- patients had three times increased normalized CD20 expression intensity (nCD20), with the intensity among CD20- B-ALL patients reaching the pretreatment nCD20 of CD20+ B-ALL patients (4.9 vs. 3.6, p = 0.666). Rituximab can be considered in managing EOI-MRD-positive CD20- B-ALL patients as the residual blasts of these patients have quantitative and qualitative increases in CD20 expression.

High-dose methotrexate pharmacokinetics and its impact on prognosis of paediatric acute lymphoblastic leukaemia patients: A population pharmacokinetic study.

This study delivers a comprehensive evaluation of the efficacy and pharmacokinetics of high-dose methotrexate (HDMTX) in a large cohort of Chinese paediatric acute lymphoblastic leukaemia patients. A total of 533 patients were included in the prognostic analysis. An association was observed between lower steady-state MTX concentrations (<56 μmol/L) and poorer outcomes in intermediate-/high-risk (IR/HR) patients. Subgroup analysis further revealed that this relationship between concentrations and prognosis was even more pronounced in patients with MLL rearrangements. In contrast, such an association did not emerge within the low-risk patient group. Additionally, utilizing population pharmacokinetic modelling (6051 concentrations from 815 patients), we identified the significant impact of physiological maturation, estimated glomerular filtration rate, sex and concurrent dasatinib administration on MTX pharmacokinetics. Simulation-based recommendations include a reduced dosage regimen for those with renal insufficiency and a specific 200 mg/kg dosage for infants under 1 year. The findings underscore the critical role of HDMTX in treating IR/HR populations and call for a reassessment of its application in lower-risk groups. An individualized pharmacokinetic dosage regimen could achieve the most optimal results, ensuring the largest proportion of steady-state concentrations within the optimal range.

Response to apheretic platelet transfusion in children of acute lymphoblastic leukemia receiving induction chemotherapy: a cross-sectional study from Bangladesh.

Disease and therapy-related hypoproliferative thrombocytopenia is a significant barrier to managing acute lymphoblastic leukaemia (ALL) patients. To reduce the risk of haemorrhage, apheretic platelet transfusion is a modern, effective, and expensive option. Since most ALL patients in Bangladesh have financial constraints, this study can shed light on the magnitude of benefit regarding the effectiveness of apheretic platelet prophylactically and therapeutically in children of ALL receiving induction chemotherapy. This observational cross-sectional study was conducted in the department of transfusion medicine and the department of paediatric haematology and oncology at a tertiary level hospital in Bangladesh from June 2020 to June 2021. A total of 33 cases of ALL were enroled in this study according to inclusion and exclusion criteria. After receiving written informed consent, relevant data were collected using a face-to-face interview with the guardian of the patients, thorough clinical examination, and relevant investigation. After the collection of all the required data, analysis was done by Stata (v.16). Mean age of the patients was 7.39±4.46 (SD), ranging from 1 to 18 years. The majority of children were aged younger than or equal to 10 years (69.70%). Male children were slightly predominant (51.5%). Significant post-transfusion platelet increment (Median pre-transfusion count 16×103/μl vs. Median post-transfusion count 133×103/μl, P<0.001) was observed. WHO bleeding grades also improved after apheretic platelet transfusion (P<0.05). Age was a significant factor associated with corrected count increment (CCI) in both univariate and multivariate analysis. In subgroup analysis, age and gender were significant predictors of CCI in therapeutic transfusion group but not in prophylactic transfusion group. Significant improvement in bleeding status and platelet count was observed following apheretic platelet transfusion.

Pretransplant Blinatumomab Improves Outcomes in B Cell Acute Lymphoblastic Leukemia Patients Who Undergo Allogeneic Hematopoietic Cell Transplantation

BackgroundBlinatumomab, a bispecific monoclonal antibody, effectively controls refractory B cell acute lymphoblastic leukemia (ALL) and promotes measurable residual disease (MRD) negativity. This study investigated the impact of pretransplant blinatumomab on allogeneic hematopoietic cell transplantation (HCT) outcomes in B cell ALL patients. MethodsWe analyzed the effect of pretransplant blinatumomab on transplant outcomes of 117 adults undergoing allogeneic HCT for B cell ALL at Princess Margaret Hospital, Toronto, between 2010 and 2021. Outcomes assessed included overall survival (OS), graft-versus-host disease and relapse-free survival (GRFS), cumulative incidences of relapse (CIR), and nonrelapse mortality (NRM). ResultsThe median follow-up was 36 months. Thirty-one participants (26.5%) received blinatumomab. Blinatumomab group had higher proportions of individuals with high disease risk index, primary induction failure and was more likely to receive dual T cell depletion with antithymocyte globulin and post-transplant cyclophosphamide. Two-year OS, GRFS, NRM, and CIR in the blinatumomab and nonblinatumomab groups were, respectively: 65.4% versus 45.6% (P = .05), 42.2% versus 17.3% (P = .01), 3.2% versus 43.0% (P = .007) and 34.4% versus 14.4% (P = .02). Blinatumomab was associated with a lower incidence of day-100 grade 2 to 4 and grade 3 to 4 acute graft-versus-host disease (aGVHD): 27.5% versus 56.7% (P = .009), and 10.9% versus 34.7% (P = .04), respectively. Multivariate analysis confirmed the association between pretransplant blinatumomab and improved OS and NRM. ConclusionsPretransplant blinatumomab is associated with improved OS and lower risk of NRM in B cell ALL patients undergoing allogeneic HCT, likely reflecting lower burden of treatment-related toxicity in this population. Larger prospective trials are warranted to validate our findings.

Involvement of the ABCB1 C3435T Variant but Not the MTHFR C677T or MTHFR A1298C Variant in High-Dose Methotrexate-Induced Toxicity in Pediatric Acute Lymphoblastic Leukemia Patients in China.

It remains unclear whether the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genetic variants are associated with methotrexate (MTX) elimination delay and high-dose MTX (HD-MTX) toxicities in the treatment of pediatric acute lymphoblastic leukemia (ALL). The aim of our study was to analyze the potential predictive role of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T in toxicities and the relationship between these variants and MTX elimination delay during HD-MTX therapy in pediatric ALL patients. We conducted a retrospective study on ALL patients receiving HD-MTX treatment with available MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genotype and 44-h plasma MTX levels. Logistic regression analyses and chi-square tests were used to assess the relationship between the variants and HD-MTX toxicities and MTX elimination delay. Genotype frequencies were in Hardy-Weinberg equilibrium. MTX elimination delay did not significantly differ between MTHFR C677T and MTHFR A1298C or ABCB1 C3435T. Leukopenia (P=0.028), neutropenia (P=0.034) and oral mucositis (P=0.023) were 6.444-fold, 4.978-fold and 9.643-fold increased, respectively, in ABCB1 C3435T homozygous genotype (TT) patients compared to wild-type (CC) patients. No significant association was found between the toxicities investigated and MTHFR C677T or MTHFR A1298C. This study showed that the ABCB1 C3435T homozygous allele genotype (TT) is associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis). These results may help to distinguish pediatric ALL patients with a relatively high risk of MTX-related toxicities before HD-MTX infusion and optimize MTX treatment.