Leukemic Blasts Research Articles

Aberrant stem cell and developmental programs in pediatric leukemia.

Hematopoiesis is a finely orchestrated process, whereby hematopoietic stem cells give rise to all mature blood cells. Crucially, they maintain the ability to self-renew and/or differentiate to replenish downstream progeny. This process starts at an embryonic stage and continues throughout the human lifespan. Blood cancers such as leukemia occur when normal hematopoiesis is disrupted, leading to uncontrolled proliferation and a block in differentiation of progenitors of a particular lineage (myeloid or lymphoid). Although normal stem cell programs are crucial for tissue homeostasis, these can be co-opted in many cancers, including leukemia. Myeloid or lymphoid leukemias often display stem cell-like properties that not only allow proliferation and survival of leukemic blasts but also enable them to escape treatments currently employed to treat patients. In addition, some leukemias, especially in children, have a fetal stem cell profile, which may reflect the developmental origins of the disease. Aberrant fetal stem cell programs necessary for leukemia maintenance are particularly attractive therapeutic targets. Understanding how hijacked stem cell programs lead to aberrant gene expression in place and time, and drive the biology of leukemia, will help us develop the best treatment strategies for patients.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

Asciminib Maintains Antibody-Dependent Cellular Cytotoxicity against Leukemic Blasts.

B cell acute lymphoblastic leukemia (B-ALL) is characterized by an accumulation of malignant precursor cells. Treatment consists of multiagent chemotherapy followed by allogeneic stem cell transplantation in high-risk patients. In addition, patients bearing the BCR-ABL1 fusion gene receive concomitant tyrosine kinase inhibitor (TKI) therapy. On the other hand, monoclonal antibody therapy is increasingly used in both clinical trials and real-world settings. The introduction of rituximab has improved the outcomes in CD20 positive cases. Other monoclonal antibodies, such as tafasitamab (anti-CD19), obinutuzumab (anti-CD20) and epratuzumab (anti-CD22) have been tested in trials (NCT05366218, NCT04920968, NCT00098839). The efficacy of monoclonal antibodies is based, at least in part, on their ability to induce antibody-dependent cellular cytotoxicity (ADCC). Combination treatments, e.g., chemotherapy and TKI, should therefore be screened for potential interference with ADCC. Here, we report on in vitro data using BCR-ABL1 positive and negative B-ALL cell lines treated with rituximab and TKI. NK cell activation, proliferation, degranulation, cytokine release and tumor cell lysis were analyzed. In contrast to ATP site inhibitors such as dasatinib and ponatinib, the novel first-in-class selective allosteric ABL myristoyl pocket (STAMP) inhibitor asciminib did not significantly impact ADCC in our settings. Our results suggest that asciminib should be considered in clinical trials.

Abstract 264: Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem cells

Abstract Background The “Differentiation therapy” has been emerging as a promising and more effective strategy against acute leukemia relapses. Objective In extension to the revolutionising therapeutic outcomes of All Trans Retinoic Acid (ATRA) to induce terminal differentiation of Acute Promyelocytic Leukemic (APL) blast cells, we decipher the potential effect of a natural compound “Esculetin” to serve as a differentiating agent in Acute Myeloid Leukemia (AML) as well as solid tumour cells. Underlaying role of Wnt signaling pathways in esculetin mediated blast cell differentiation was also evaluated. Methods Human acute myeloid leukemic cells (Kasumi-1) with t(8;21/AML-ETO) translocation were used as a model system. Growth inhibitory and cytotoxic activity of esculetin were analysed using growth kinetics and MTT assay. Morphological alterations, cell scatter characteristics, NBT reduction assay and cell surface marker expression pat- terns were analysed to detect terminally differentiated phenotypes. We employed RT2profiler PCR array system for the analysis of transcriptome profile of Wnt signaling components. Calcium inhibitors (TMB8 and Amlodipine) and Transforming growth factor beta (TGF-β) were used to modulate the Wnt signaling axes. To investigate whether esculetin exerts the similar effects on the invasive solid tumour cancer stem cells subset, we used a cellular model system of colon carcinoma HCT116 cells reflecting EMT phenotype. Results We illustrate cytotoxic as well as blast cell differentiation potential of esculetin on Kasumi-1 cells. Morphological alterations akin to neutrophilic differentiation as well as the corresponding acquisition of myeloid lineage markers indicate terminal differentiation potential of esculetin in leukemic blast cells. Exposure to esculetin also resulted in downregulation of canonical Wnt axis while upto ~ 21 fold upregulation of non-canonical axis associated genes. Esculetin also showed potential to revert the CSC marker expressions and concurrent EMT consistent with reduced functional aggressiveness in colon cancer cells. Conclusions Our study highlights the importance of selective use of calcium pools as well as “axis shift” of the canonical to non-canonical Wnt signaling upon esculetin treatment which might abrogate the inherent proliferation to release maturation arrest and induce the differentiation in leukemic blast cells as well as CSC stem cells. The current findings provide further therapeutic interventions to consider esculetin as a potent differentiating agent to counteract relapses. Citation Format: Ankit Mathur, Daman Saluja. Differentiation therapy: Esculetin as a potent agent to alter cellular plasticity of leukemic & solid tumor stem 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 264.

Abstract 629: Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem cells

Abstract Introduction: In patients with acute myeloid leukemia (AML), CD123 has been shown to have high expression on blast cells and leukemic stem cells (LSC) compared with normal hematopoietic stem cells (HSCs) and other more mature CD34+ subsets (Zhou J, World J Stem Cells 2014). LSCs are inherently resistant to standard of care chemotherapeutics and LSC persistence after chemotherapy is associated with disease relapse. VIP943 is an ADC consisting of an anti-CD123 antibody, a unique linker cleaved intracellularly by legumain which is required for release, and activation of a novel kinesin spindle protein inhibitor (KSPi) payload that accumulates inside the cell. Herein, we evaluated the potential of VIP943 to target LSC and progenitor cells. Methods: Cytotoxicity assay: Bone marrow aspirates (BMA) and peripheral blood from previously untreated patients with primary or transformed from myelodysplastic syndrome AML were used in this experiment. Cells were treated with VIP943 (66 nM or 330 nM). After the end of the 48 h or 72 h incubation, the CD123+ cells and the CD34+CD38- LSC were detected by flow cytometry. Characterization of progenitor cell population: Fresh BM samples from healthy volunteers (HVBM) were stained with specific monoclonal antibodies to identify progenitor cell populations. Depletion assay: The effect of VIP943 on HVBM was evaluated by measuring the depletion of CD34+ progenitor cell populations in comparison to the anti-CD33-ADC, gemtuzumab ozogamicin (gem-oz). At the end of the incubation time, red blood cells were lysed; the remaining cells were stained with a cocktail of antibodies to discriminate between progenitor cells and analyzed by flow cytometry. Results: Treatment of patient-derived CD123+ leukemic blasts with VIP943 resulted in a 50% reduction after 48 h and up to 80% at 72 h. A reduction of >70% of CD123+CD34+CD38- LSCs derived from BM aspirates of untreated patients with AML was achieved after incubation with VIP943 up to 72h. In addition, HVBM samples derived from five healthy volunteers were treated with VIP943 or gem-oz in a depletion assay, where gem-oz was toxic to CD34+ cells with an EC50 of 0.16 µM in contrast to VIP943 with an EC50 value of 8.83 µM. Conclusions: In this in vitro analysis of primary samples from patients with AML, activity of VIP943 is observed in AML patient-derived CD123+ blasts as well as chemoresistant LSCs. At anticipated pharmacologic levels, VIP943 shows no adverse effects on HSCs unlike gem-oz suggesting an improved therapeutic index. In the ongoing first-in-human dose-escalation study in subjects with advanced CD123+ hematologic malignancies, VIP943 demonstrates a promising safety profile (NCT06034275). Citation Format: Beatrix Stelte-Ludwig, Tibor Schomber, Melanie M. Frigault, Joseph Birkett, Amy J. Johnson, Anne-Sophie Rebstock, Sebastian Ludwig, Raquel Izumi, Ahmed Hamdy. Activity of VIP943 on AML patient-derived leukemic blasts and healthy donor-derived bone marrow hematopoietic stem 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 629.

Abstract 7397: Application of state transition theory for predicting responses of acute myeloid leukemia to chemotherapy

Abstract Acute Myeloid Leukemia (AML) is aggressive cancer that initiates in the bone marrow (BM) and circulates in the peripheral blood (PB). The AML initiation and progressions are complex disease evolution associated with gene mutations and chromosomal abnormalities that change expression profiles of downstream genes characterizing functional networks of leukemic cells. We previously reported the application of the state-transition theory to characterize AML disease progression by modeling transcriptome changes during AML progression as a dynamic system that undergoes state-transition in an AML state-space characterized by an AML potential. Here, we investigate the impact of treatment on transcriptome state-transition and examine the hypothesis that treatment dynamically alters the AML potential. We use the Cbfb-MYH11 (CM) knock-in AML mouse model and a ”5+3” chemotherapy regimen to model the impact of current standard of care for newly diagnosed AML. CM leukemic mice were treated with a combination of cytarabine (Ara-C; 50mg/kg/day; 5 days) and Daunorubicin (DNR; 1.5mg/kg/day; 3 days) after detection of overt leukemia, defined by > 20% leukemia blast (cKit+) detected by flow cytometry in the PB. Time-series samples of peripheral blood mononuclear cell (PBMC) (n=174) were collected weekly before, during, and following “5+3” chemotherapy and subjected to RNA sequencing. All samples were mapped to the AML state-space constructed based on time-sequential RNA-seq collected over the course of CM AML development. Based on the state-transition model, all 10 treated AML mice achieved partial response to chemotherapy with a mean time to relapse (return to leukemia state) of 5 weeks confirming the anti-leukemia effects of treatment by the blast percentages (ckit%). We used the state-transition treatment model to study the dynamics of chemotherapy on the AML potential using state-space trajectories before, during, and after “5+3” chemotherapy. Dynamic changes of concentrations of Ara-C and DNR in the CM mouse model during ”5+3” chemotherapy were modeled by drug doses, half-life, and Heaviside step function. The drug effects were introduced to the AML potential as anti-leukemic treatment forces. Several treatment protocols were examined to find combinations of drug doses and timing that showed the longest survival rates. The model predicted that sequential doses reducing 50% of the original “5+3” chemotherapy dose would produce the longest survival rate. The model suggests that controlling the state-space trajectories in the healthy state with reduced doses will provide the most effective outcomes from “5+3” chemotherapy. Citation Format: Lisa Uechi, Yu-Hsuan Fu, David E. Frankhouser, Lianjun Zhang, Ying-Chieh Chen, Denis O'Meally, Sergio Branciamore, Jihyun Irizarry, Bin Zhang, Guido Marcucci, Ya-Huei Kuo, Russell C. Rockne. Application of state transition theory for predicting responses of acute myeloid leukemia to chemotherapy [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 7397.

Abstract 3855: Kinetics of neoantigen-specific T cells in post-transplant relapse

Abstract Introduction: Graft-versus-leukemia effect (GVL) underpins the curative mechanism of allogeneic stem cell transplantation (alloSCT) for acute leukemias. Donor-derived T cells can recognize various antigens, including human leukocyte antigen (HLA), minor-histocompatibility antigens (miHA), over-expressed antigens, or neoantigens derived from somatic mutations of leukemia. However, the role of neoantigens has not been fully investigated in GVL. Here, we aimed to predict neoantigens and identify neoantigen-reactive T cells using samples after alloSCT. Materials: Leukemia blasts were purified from relapsed samples. Recipient saliva or T cells from pre-transplant peripheral blood (PB) were used as a recipient germline control, while donor monocyte or CD34 cells were used as donor controls. DNA/RNA isolated from these specimens were analyzed for whole exome sequencing (WES) paired with RNAseq. Somatic mutations were called using blasts and recipient germline controls by mutect and mpileup and annotation with SnpEff. Somatic mutations with variant allele frequency >20% were screened for neoantigen prediction bound to recipient HLA (IC50 < 500nM) by pVAC-seq. We also genotyped 73 SNPs associated with publicly known miHAs. Serial PB were stained for HLA-A*02:01 tetramers loaded with neoantigens or CMVpp65. T cells were stimulated with neoantigens for 10 days and tested for cytokine production in responses to target or control peptides. Results: We screened neoantigens in 8 patients relapsing after alloSCT (7 acute myeloid leukemia and 1 acute lymphoblastic leukemia) and their donors (4 matched siblings, 1 matched unrelated, 3 haploidentical). In 7 subjects, WES/RNAseq showed an average of 273 somatic mutations per patient (range 108- 609). In one subject, targeted next-generation sequencing was used to detect 2 somatic mutations. Among these mutations, seven out of eight subjects (87.5%) had potential neoantigen candidates, with a median number of 9 antigens per subject. In contrast, only 2 subjects (25%) had potential miHA. UPN8, a responder to donor lymphocyte infusion (DLI), showed dominant CMVpp65-specific T cells at the time of relapse and emergence of TP53- (neoantigen) or HA-2- (miHA) specific T cells after relapse. HA-2-specific T cells upregulated PD-1 expression at the time of graft-versus-host disease (GVHD), indicating activation of these miHA-specific T cells during GVHD. In two subjects (UPN3 and UPN8) who achieved long-term remission after DLI, CD8 T cells specific to neoantigens were detectable by IFN-γ/CD107a production in reaction to mutant peptides (SDE2.pK123X or TP53.pR248W) from post-relapse samples. Discussion: Our study showed the feasibility of in-silico neoantigens prediction and neoantigen-specific T-cell detection from a subject who developed relapse after alloSCT. Further investigation is needed to test if neoepitope burden and miHA disparity could correlate with transplant outcomes. Citation Format: Biswas Neupane, Jui-En Ray Lee, Kedwin Ventura, Kayla Parr, Kevin Quann, Sawa Ito. Kinetics of neoantigen-specific T cells in post-transplant relapse [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 3855.

Abstract 7011: The DNA methylation landscape across the TCR loci genes in patients with acute myeloid leukemia

Abstract The T-cell receptor (TCR) repertoire is partly shaped by epigenetic modifications, including DNA methylation. Hypomethylating agents like decitabine are used to treat patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The precise DNA methylation patterns within the TCR loci and their dynamics associated with HMAs treatments remain underexplored. Here, we analyzed methylation patterns across TCR alpha and beta gene loci in T cells from GSE175758 and GSE67170 datasets, comparing patterns between patients with AML and healthy controls. We also investigated the impact of decitabine on the methylation patterns of TCR gene loci in patients with AML and MDS (GSE175758 and GSE80762).The overall levels of methylation between T cells from healthy donors and patients with AML were significantly different within the TRAV (0.55 vs 0.42, P < 0.001) and TRBV regions (0.70 vs 0.59, P < 0.001) with 46 TRAV and 33 TRBV sites differentially methylated positions, respectively. However, methylation patterns in HSCs (GSE63409), T cells from healthy donors and patients with AML and those in AML cells were strongly correlated (R > 0.67, range 0.67-0.99, P < 0.001).Because decitabine preferentially acts on leukemic cells and given the high correlation levels between AML cells and T cells across datasets we analyzed the effects of decitabine on TCR DNA methylation patterns in AML cells. In the leukemic cells decitabine treatment led to significant demethylation across 33 and 30 CpG sites within the TRAV and TRBV genes, respectively. In the pre-treatment AML samples higher methylation beta values were observed in differentially methylated positions (DMPs) compared with non-DMPs (TRAV: 0.847 vs 0.407; P < 0.001; TRBV: 0.771 vs 0.538; P = 0.049). In samples largely enriched of myeloid leukemic blasts, we found that TRAV and TRBV loci methylation patterns differ significantly between AML patients with good, moderate, and poor risk patients (P < 0.001). We found TRBV6-1 position 1 and 2 methylation was lower in patients with good risk, compared with intermediate risk (pos1: 0.07 vs 0.13, P=0.04; pos2: 0.1 vs 0.2, P = 0.002) and poor risk (pos 1: 0.07 vs 0.21, P < 0.001; pos 2: 0.1 vs 0.32, P < 0.001) and was lower in patients with intermediate risk compared with poor risk (pos1: 0.13 vs 0.21, p=0.007; pos 2: 0.21 vs 0.32, P = 0.001). The high correlation of TCR DNA methylation patterns between the T cells and the myeloid cells, suggests the need for similar analyses in the context of AML T cells. The study highlights the presence of a conserved TCR loci methylated signatures which is highly correlated between myeloid leukemic cells and T cells and is associated with clinical outcomes, offering a potential avenue for therapeutic intervention. Hypomethylating agents, such as decitabine, can modulate TCR loci methylation patterns. Citation Format: Mateusz Pospiech, John Beckford, Advaith M. Kumar, Mukund Tamizharasan, Jaqueline Brito, Gangning Liang, Serghei Mangul, Houda Alachkar. The DNA methylation landscape across the TCR loci genes in patients with acute myeloid 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 7011.

Abstract 2548: Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples

Abstract Background: Assessing drug efficacy in primary acute myeloid leukemia (AML) patient cells is crucial for pinpointing drug targets and rationally selecting therapies for patients. We have previously shown that our flow cytometry-based predictive precision medicine platform (PPMP) predicts patient outcomes for standard-of-care (SOC) AML treatments using fresh blood or bone marrow samples with high precision. Here, we wanted to test the feasibility and efficacy of the PPMP on cryopreserved patient-derived leukemic samples. However, cryopreserved leukemic samples are known for exhibiting low viability and cell recovery, rendering their practical utilization often challenging. In this study, we adapt an existing methodology for cryopreservation for patient-derived leukemic cells to investigate the potential of cryopreserved samples as an alternative to fresh samples for ex vivo prediction of treatment outcomes on our PPMP. Methods: We screened freshly isolated white blood cells from AML patients on the PPMP to assess their response to multiple SOC single agents and combinations. The assay was also run without drug treatments immediately after receiving the fresh sample (≤ 2 days from draw) to establish a phenotype baseline. The cryopreserved samples were processed using a similar protocol, with post-thaw culture times of either 24 or 48 hours in two types of cytokine-containing media. A comparative analysis between untreated fresh and cryopreserved samples determined the optimal post-thaw culture time for cryopreserved samples. Once culture time was optimized, we screened cryopreserved samples on the PPMP with identical SOC drug conditions as the fresh samples and compared the response of leukemic blasts to drug treatment as well as impacts on other phenotypic markers. Results: Based on our preliminary data, we found that culturing cryopreserved cells for 24 hours post-thaw was sufficient to maintain a minimum cell viability of 80% while closely maintaining a phenotype that recapitulated that of the corresponding fresh samples. In response to drug treatments within the ex vivo assays, preliminary data support a correlation (Decitabine single agent, R2=0.81) between the response profiles of cryopreserved and fresh patient samples as gauged from AUC calculations. Conclusion: While the preferred method for screening is to use fresh patient samples, many practical constraints make it challenging or unfeasible. The ability of Notable Lab’s high-throughput platform to use cryopreserved samples opens the doors to enhanced retrospective studies leveraging clinically annotated data from biobanks or clinical trials. These retrospective studies provide a substantial reduction in the time needed to conclude such investigations. Citation Format: Lokesh Patil, Christine J. Gu, Wade Anderson, Nicola Long, Kara Johnson, Cristina Tognon, Markus D. Lacher. Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples [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 2548.

Abstract 3952: Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell acute lymphoblastic leukemia

Abstract Detection of minimal residual disease (MRD) after initial diagnosis (IDX) and therapy is a strong prognostic marker for relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have explored clonal genetic and gene expression changes between initial diagnosis and relapse. However, studying tumor cells present at MRD is challenging due to their low abundance. Examining the genomic profiles of leukemia cells that resist induction chemotherapy and understanding their evolutionary relationship to cells present at diagnosis may lead to a better understanding of the cells that eventually seed relapse. We analyzed somatic point mutations, indels, and copy number variants from 41 pediatric B-ALL patients with high-risk B-ALL. We specifically focused on patients from the following four common high-risk subtypes: KMT2A-rearranged, Ph+, Ph-like, and iAMP21, who had detectable disease at the end of induction. Leukemic burden at end-induction (day 29) ranged from 0.03% to 66.00%. Data were generated in partnership with the National Cancer Institute’s Human Tumor Atlas Network and the Children’s Oncology Group. Our data set of 41 patients includes 39 with paired initial diagnostic and end-induction samples that we used to create tumor evolution models based on single cells across disease stages. We produced single-cell multiome gene expression (scRNA) and chromatin accessibility (scATAC) profiles (10x Genomics) with a median of 7428 leukemic blast cells at IDX (range: 2490-14219) and 1907 at MRD (range: 240-10167) after filtering. We called somatic mutations from scRNA and scATAC using SComatic and Monopogen, and we detected copy number changes using Numbat and EpiAneufinder. We also designed a targeted DNA sequencing panel including 335 variants based on B-ALL literature and database searches to produce scDNA (Mission Bio) for 24 patients (16 IDX-MRD pairs) with a median of 2346 cells profiled at IDX (range: 340-8849) and 1828 at MRD (range: 596-5133). We built tumor phylogenies based on somatic SNVs and CNVs with COMPASS. Using multiple tools and complementary single-cell omics data types to detect somatic changes improved our confidence in the events used for tumor evolution tree construction. Our approach allowed us to define recurrent genomic events and patterns across pediatric B-ALL subtypes and to describe the clonal evolution of cells measured at initial diagnostic and end-induction disease stages. We were also able to link cell-specific mutation events to their transcriptional and epigenomic signatures, including mapping mutation events along the B-cell development trajectory and within differentially expressed gene pathways. Some cases showed dramatic shifts in subclonal structure between IDX and MRD that may be related to therapy-induced clonal selection, such as a loss of NRAS hotspot mutation. Citation Format: Steven M. Foltz, Avi Loren, Changya Chen, Rushabh Mehta, Elizabeth Li, Jason Xu, Fatemeh Alikarami, Kathrin M. Bernt, Kai Tan. Multiomic single-cell tumor evolution models of minimal residual disease in pediatric B-cell 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 3952.

CD7-positive leukemic blasts with DNMT3A mutations predict poor prognosis in patients with acute myeloid leukemia.

DNMT3A mutations can be detected in premalignant hematopoietic stem cells and are primarily associated with clonal hematopoiesis of indeterminate potential; however, current evidence does not support assigning them to a distinct European Leukemia Net (ELN) prognostic risk stratification. CD7 is a lymphoid antigen expressed on blasts in approximately 30% of acute myeloid leukemia (AML), and its role in AML remains unclear and depends on subgroup evaluation. This study investigated the prognostic value of DNMT3A mutation combined with CD7 expression in AML. We retrospectively analyzed the clinical data of 297 newly diagnosed non-M3 AML patients. According to the DNMT3A mutation and CD7 expression in AML cells, patients were divided into the DNMT3A-mutated/CD7-positive (CD7+), DNMT3A-mutated/CD7-negative (CD7-), DNMT3A-wild-type/CD7+, and DNMT3A-wild-type/CD7- groups. The DNMT3A-mutated/CD7+ group had lower complete remission rates and higher relapse rates. Importantly, these patients had significantly shorter overall survival (OS) and relapse-free survival (RFS). Furthermore, multivariate analysis showed that CD7+ with DNMT3A mutation was an independent risk factor for OS and RFS. CD7+ with DNMT3A mutation predicts a poor prognosis in AML patients, and the immunophenotype combined with molecular genetic markers can help to further refine the current risk stratification of AML.

Laboratory characterization of the pediatric B/T subtype of mixed-phenotype acute leukemia: Report of a case series.

Mixed-phenotype acute leukemia (MPAL) is a rare disease associated with difficulties in the correct lineage assignment of leukemic cells. One of the least common subtypes within this category is characterized by the simultaneous presence of B- and T-lineage-defining antigens. Each case of suspected B/T MPAL should be considered in light of all available laboratory and clinical data to avoid misdiagnosis. In this study, we describe 6 pediatric patients who presented with leukemic blasts bearing B- and T-lineage antigens at diagnosis, including their clinical, immunophenotypic, morphologic, and cytogenetic characteristics. In 3 patients, more or less distinct populations of B- and T-lymphoid origin were found; the other 3 patients had a single mixed-phenotype blast population. All cases fulfilled the World Health Organization criteria, but not all of them turned out to be bona fide cases of B/T MPAL according to the available clinical and laboratory data. Found genetic lesions were helpful for the confirmation of MPAL instead of 2 concomitant tumors, but for a general B/T MPAL diagnosis, genetic studies provided the only descriptive data. The accurate diagnosis of B/T MPAL requires a multidisciplinary approach combining high-tech laboratory methods and close cooperation between treating physicians and pathologists.

White blood cell count nadir to zero following intensive chemotherapy as a predictive factor for patients with acute myeloid leukemia

Predictors for response to intensive therapy in AML have focused on baseline factors: percent leukemic blasts in marrow, cytogenetic/molecular genetic abnormalities, and presence of secondary AML. Non-baseline dynamic factors, occurring after induction but before response, may be useful for decisions related to salvage chemotherapy. We hypothesized white blood cell (WBC) count nadir after induction may be a real time indicator of treatment efficacy. We also examined whether time to stem cell transplant (SCT) or baseline molecular genetic abnormalities are associated with a low nadir. Data showed WBC nadir = 0 was a negative predictor for response to intensive induction and was correlated with reduced overall survival and progression free survival. Patients with WBC nadir = 0 did not have a significantly longer time to SCT, and none of the mutations increased the likelihood of reaching WBC nadir = 0. WBC nadir may be a useful real-time monitor in AML patients receiving intensive induction chemotherapy.

Continuous 72-h infusion of zosuquidar with chemotherapy in patients with newly diagnosed acute myeloid leukemia stratified for leukemic blast P-glycoprotein phenotype.

To evaluate the safety, tolerability, pharmacodynamics (PD), and potential efficacy of zosuquidar (Zos) in combination with daunorubicin and cytarabine in elderly patients with newly diagnosed acute myeloid leukemia (AML). Patients with AML (N = 106) were treated with Zos as a 72-h continuous intravenous (CIV) infusion along with chemotherapy. Leukemic blasts from the patients were assessed for P-glycoprotein (P-gp) function using ex vivo bioassays for screening and PD analyses. Patient outcomes were categorized according to primary (N = 56) and secondary (N = 50) AML cohorts (pAML and sAML, respectively) and stratified into P-gp-high and P-gp-low subgroups. Patients with P-gp-high blasts exhibited comparable overall remission rates (ORR) to those with P-gp-low blasts in both the pAML and sAML cohorts. The P-gp-high and P-gp-low subgroups in the pAML cohort exhibited similar overall survival (OS). Patients with sAML and P-gp-high blasts exhibited significantly better OS than those in the P-gp-low subgroup. PD analyses revealed that Zos infusion provided 82h of uninterrupted effective ≥ 90% inhibition of P-gp functional activity in leukemic blasts. These observations provide evidence of Zos efficacy with the 72-h CIV infusion approach. The similarity of ORR in the P-gp-high and P-gp-low subgroups is consistent with Zos-mediated neutralization of P-gp as verified by PD analyses. The bioassay identified sAML patients most likely to respond favorably to Zos co-therapy indicating feasibility as a Zos companion diagnostic. A follow-up placebo-controlled trial is needed to verify these promising results. NCT00129168; First posted on August 11, 2005.

Flow cytometric detection of leukemic blasts in Libyan pediatric patients with acute lymphoblastic leukemia

ABSTRACT The diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

When inflammatory stressors dramatically change, disease phenotypes may transform between autoimmune hematopoietic failure and myeloid neoplasms.

Aplastic anemia (AA) and hypoplastic myelodysplastic syndrome are paradigms of autoimmune hematopoietic failure (AHF). Myelodysplastic syndrome and acute myeloid leukemia are unequivocal myeloid neoplasms (MNs). Currently, AA is also known to be a clonal hematological disease. Genetic aberrations typically observed in MNs are detected in approximately one-third of AA patients. In AA patients harboring MN-related genetic aberrations, a poor response to immunosuppressive therapy (IST) and an increased risk of transformation to MNs occurring either naturally or after IST are predicted. Approximately 10%-15% of patients with severe AA transform the disease phenotype to MNs following IST, and in some patients, leukemic transformation emerges during or shortly after IST. Phenotypic transformations between AHF and MNs can occur reciprocally. A fraction of advanced MN patients experience an aplastic crisis during which leukemic blasts are repressed. The switch that shapes the disease phenotype is a change in the strength of extramedullary inflammation. Both AHF and MNs have an immune-active bone marrow (BM) environment (BME). In AHF patients, an inflamed BME can be evoked by infiltrated immune cells targeting neoplastic molecules, which contributes to the BM-specific autoimmune impairment. Autoimmune responses in AHF may represent an antileukemic mechanism, and inflammatory stressors strengthen antileukemic immunity, at least in a significant proportion of patients who have MN-related genetic aberrations. During active inflammatory episodes, normal and leukemic hematopoieses are suppressed, which leads to the occurrence of aplastic cytopenia and leukemic cell regression. The successful treatment of underlying infections mitigates inflammatory stress-related antileukemic activities and promotes the penetration of leukemic hematopoiesis. The effect of IST is similar to that of treating underlying infections. Investigating inflammatory stress-powered antileukemic immunity is highly important in theoretical studies and clinical practice, especially given the wide application of immune-activating agents and immune checkpoint inhibitors in the treatment of hematological neoplasms.

Significance of OCT3/4 and SOX2 antigens expression by leukemic blast cells in adult acute leukemia

ObjectiveThis study aimed to address the prognostic impact of SOX2 and OCT3/4 expression on adult acute leukemia patients’ outcomes.MethodsSOX2 and OCT3/4 expression by blast cells were evaluated by flow cytometry in 80 acute leukemia patients and 8 healthy controls.ResultsBaseline SOX2 and OCT3/4 expression were significantly higher in both ALL (P = < 0.001, P = 0.005 respectively) and AML patients (P < 0.001, P = 0.003 respectively) as compared to control, and decline at complete remission (CR) and elevated again at relapse. High SOX2 and OCT3/4 levels were significantly correlated with the presence of adverse risk stratification parameters.ConclusionOur findings indicated that both SOX2 and OCT3/4 could serve as biomarkers that could improve risk stratification of acute leukemia patients. Also, both SOX2 and OCT3/4 might be a therapeutic target, especially in resistant acute leukemia.

Single agent subcutaneous blinatumomab for advanced acute lymphoblastic leukemia.

Blinatumomab is a BiTE® (bispecific T-cell engager) molecule that redirects CD3+ T-cells to engage and lyse CD19+ target cells. Here we demonstrate that subcutaneous (SC) blinatumomab can provide high efficacy and greater convenience of administration. In the expansion phase of a multi-institutional phase 1b trial (ClinicalTrials.gov, NCT04521231), heavily pretreated adults with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) received SC blinatumomab at two doses: (1) 250 μg once daily (QD) for week 1 and 500 μg three times weekly (TIW) thereafter (250 μg/500 μg) or (2) 500 μg QD for week 1 and 1000 μg TIW thereafter (500 μg/1000 μg). The primary endpoint was complete remission/complete remission with partial hematologic recovery (CR/CRh) within two cycles. At the data cutoff of September 15, 2023, 29 patients were treated: 14 at the 250 μg/500 μg dose and 13 at 500 μg/1000 μg dose. Data from two ineligible patients were excluded. At the end of two cycles, 12 of 14 patients (85.7%) from the 250 μg/500 μg dose achieved CR/CRh of which nine patients (75.0%) were negative for measurable residual disease (MRD; <10-4 leukemic blasts). At the 500 μg/1000 μg dose, 12 of 13 patients (92.3%) achieved CR/CRh; all 12 patients (100.0%) were MRD-negative. No treatment-related grade 4 cytokine release syndrome (CRS) or neurologic events (NEs) were reported. SC injections were well tolerated and all treatment-related grade 3 CRS and NEs responded to standard-of-care management, interruption, or discontinuation. Treatment with SC blinatumomab resulted in high efficacy, with high MRD-negativity rates and acceptable safety profile in heavily pretreated adults with R/R B-ALL.

Incidence, risk factors, and outcome of asymptomatic central nervous system involvement in adult patients with acute myeloid leukemia.

Examination of central nervous system (CNS) involvement is not routine diagnostic practice in adult patients with acute myeloid leukemia (AML). Therefore, many asymptomatic patients with CNS involvement might go undetected. The effect of CNS involvement on the AML disease course is not well defined, with conflicting results regarding clinical outcome. This study aimed to determine the incidence of asymptomatic CNS involvement in AML estimated by multiparametric flow cytometry of cerebrospinal fluid (MFC-CSF) at diagnosis, the related potential risk factors, and prognosis. In total, 645 patients with de novo AML were screened; 183 (28.4%) of them fulfilled institutional practice for MFC-CSF analysis based on presence of CNS symptoms and/or clinical features. CNS symptoms and signs were observed in 8/183 (4.4%) patients, but most patients (175/183, 95.6%) were asymptomatic. In the asymptomatic group, 73/175 (41.7%) patients had positive or suspicious cerebrospinal fluid (CSF) findings categorized as CNS positive (CNSpos) and 102/175 (58.3%) had normal CNS findings categorized as CNS negative (CNSneg). The presence of leukemic blasts was confirmed in 81/183 (44.3%) patients; the total incidence of CNS involvement in the whole AML group was 12.6% (81/645). Compared with asymptomatic patients with CNSneg, those with CNSpos had a significantly higher frequency of lymphadenopathy, white blood cell count ≥30×109/L, presence of the monocytic phenotype, and a high percentage of bone marrow (BM) blasts. The multivariate logistic regression model identified monocytic phenotype (p=0.047) and high percentage of BM blasts (p=0.042) as predictors for CNSpos. CNSpos did not affect overall survival in patients with AML. There was a higher incidence of CNS involvement in asymptomatic adult patients with de novo AML, emphasizing possible undervalued rates of CNS disease at diagnosis. Prospective studies should determine whether diagnostic lumbar puncture for MFC-CSF analysis and CNS prophylaxis could contribute to better selection and prognosis in this patient population.

Monosomy 7/del(7q) cause sensitivity to inhibitors of nicotinamide phosphoribosyltransferase in acute myeloid leukemia

AbstractMonosomy 7 and del(7q) (-7/-7q) are frequent chromosomal abnormalities detected in up to 10% of patients with acute myeloid leukemia (AML). Despite unfavorable treatment outcomes, no approved targeted therapies exist for patients with -7/-7q. Therefore, we aimed to identify novel vulnerabilities. Through an analysis of data from ex vivo drug screens of 114 primary AML samples, we discovered that -7/-7q AML cells are highly sensitive to the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is the rate-limiting enzyme in the nicotinamide adenine dinucleotide salvage pathway. Mechanistically, the NAMPT gene is located at 7q22.3, and deletion of 1 copy due to -7/-7q results in NAMPT haploinsufficiency, leading to reduced expression and a therapeutically targetable vulnerability to the inhibition of NAMPT. Our results show that in -7/-7q AML, differentiated CD34+CD38+ myeloblasts are more sensitive to the inhibition of NAMPT than less differentiated CD34+CD38– myeloblasts. Furthermore, the combination of the BCL2 inhibitor venetoclax and the NAMPT inhibitor KPT-9274 resulted in the death of significantly more leukemic blasts in AML samples with -7/-7q than the NAMPT inhibitor alone. In conclusion, our findings demonstrate that AML with -7/-7q is highly sensitive to NAMPT inhibition, suggesting that NAMPT inhibitors have the potential to be an effective targeted therapy for patients with monosomy 7 or del(7q).