Cell Precursor Acute Lymphoblastic Leukemia Research Articles

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

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

Prognostic significance of Wilms' tumor gene 1 expression in children with B-cell precursor acute lymphoblastic leukemia.

The prognostic role of Wilms' tumor 1 (WT1) gene expression at diagnosis in children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is still controversial. We detected the WT1 transcript levels of 533 de novo pediatric BCP-ALL patients using TaqMan-based real-time quantitative PCR and analyzed their clinical features. The WT1 transcript levels differed among the distinct molecularly defined groups, with the highest levels in the KMT2A rearrangements (KMT2A-r) group. According to the results of the X-tile software, all patients were divided into two groups: WT1/ABL ≥ 0.24% (group A) and <0.24% (group B). The proportions of patients whose age was ≥10 years old, with immunophenotype of Pro-B, belonging in high-risk group, or with minimal residual disease (MRD) ≥ 0.01% at week 12 were significantly higher in group A than in group B. In the B-other group, WT1 overexpression was an independent risk factor of overall survival (OS) rate (P = 0.042), and higher MRD ≥ 0.01% at week 12 was associated with lower OS rate (P<0.001) and event-free survival rate (P<0.001). Moreover, the subgroup analysis revealed that, in patients with initial WBC<50 × 109/L or MRD<0.1% at day 33 or MRD<0.01% at week 12 or in the standard-risk group, WT1 overexpression led to a poorer outcome in comparison with those with WT1 downexpression (P<0.05). Therefore, pediatric BCP-ALL with WT1 overexpression had unique clinico-pathological characteristics and poor treatment response. In B-other patients, WT1 overexpression at diagnosis predicted an inferior prognosis. The WT1 gene may serve as a biomarker for monitoring residual disease in the B-other population, especially in children in the standard-risk group.

Chinese expert consensus on diagnosis and treatment of adult early T cell precursor acute lymphoblastic leukemia (2023)

Chinese expert consensus on diagnosis and treatment of adult early T cell precursor acute lymphoblastic leukemia (2023)

PELI2 regulates early B-cell progenitor differentiation and related leukemia via the IL-7R expression.

Little is known about the transition mechanisms that govern early lymphoid lineage progenitors from common lymphoid progenitors (CLP). Pellino2 (PELI2) is a newly discovered E3 ubiquitin ligase, which plays important roles in inflammation and the immune system. However, the physiological and molecular roles of PELI2 in the differentiation of immune cells are largely unknown. Here, by using a conditional knockout mouse model, we demonstrated that PELI2 is required for early B-cell development and stressed hematopoiesis. PELI2 interacted with and stabilized PU.1 via K63-polyubiquitination to regulate IL-7R expression. The defects of B-cell development induced by PELI2 deletion were restored by overexpression of PU.1. Similarly, PELI2 promoted TCF3 protein stability via K63-polyubiquitination to regulate IL-7R expression, which is required for the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. These results underscore the significance of PELI2 in both normal B lymphopoiesis and malignant B-cell acute lymphoblastic leukemia via the regulation of IL-7R expression, providing a potential therapeutic approach for BCP-ALL.

N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells

Today, the majority of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) patients survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell permeable cAMP analogue 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs.. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

In Vitro Efficacy of Anti-HER2 CAR T-Cells in HER2 + BCP-ALL

Introduction: Around 30% of adults and 15% of children with B cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse after a first line of treatment. The prognosis of these patients remains poor. Recent advances have involved targeted therapies with monoclonal antibodies and chimeric antigen receptor (CAR) T-lymphocytes (anti-CD19 CAR T-cells), which have produced very encouraging results in the high risk BCP-ALL setting(Maude et al. 2018). Nevertheless, half of these patients relapse after such therapies. In a third of cases, the disease no longer expresses the antigen previously targeted. New targets are therefore needed. HER2 is expressed in above one third of adult BCP-ALL (Chevallier et al. 2004) and 10% of childhood BCP-ALL (Camuset et al. 2021). HER2 expression is associated with chemoresistance and poor prognosis (Chevallier et al. 2004) (Camuset et al. 2021). The anti-HER2 antibody trastuzumab was prospectively tested in adult patients with HER2+ BCP-ALL, but yielded disappointing results (Chevallier et al. 2012). Anti-HER2 CAR T-cells could be an interesting alternative. Indeed, they have shown encouraging preclinical efficacy in solid tumors expressing HER2/neu (Budi et al. 2022). The present work was designed to evaluate, in vitro, the sensitivity of HER2+ BCP-ALL to anti-HER2 CAR engineered Epstein Barr virus (EBV) specific T-cells. Methods: DMSO-frozen cell-samples from 6 HER2+ BCP-ALL patients were available for this study. Patient characteristics are shown in Table 1. Blast cells from patient samples were preferred to cell lines in order to be more representative of BCP-ALL biology. level of HER2 expression on BCP-ALL samples was determined by flow cytometry and compared to the HER2 expressing breast-cancer cell line BT474. Anti-EBV cytotoxic T lymphocytes (CTLs), armed with a trastuzumab-based scFv-CH2-CH3-IgG2A-FcεRIγ chimeric receptor as previously described (Clémenceau et al. 2015) were used for long-term cytoxicity assays. For 4 samples, non-transduced anti-EBV CTLs were used as controls. Cytotoxic activity was assessed at a 3-to-1 effector-to-target ratio, in a flow cytometry assay. Cells were harvested at H0, H4 and H24 and stained with a viability stain (VS780). Further staining with anti-CD22 or anti-CD24 (depending on the BCP-ALL immunophenotype) and anti-CD3 antibodies allowed to characterize blast cells and CAR T-cells respectively. The study was approved by the local review board and two alive patients provided informed consent. Results: The level of HER2 expression by the 6 HER2+ BCP-ALL samples was variable with a median relative fluorescence intensity ratio (RFI) of 9 (range 1.8 to 16). These levels of expression were lower than those of the BT474 cell line (RFI= 79). No BCP-ALL lysis was observed when cells were incubated with trastuzumab alone (not shown). When co-cultured with non-transduced anti-EBV CTLs (CAR negative) (n=4), a decrease of residual viable BCP-ALL cells was observed at variable levels (Figure 1A). The lysis observed at 24h was possibly due to allogeneic recognition since anti-EBV CTLs were not autologous to the leukemias tested. Conclusion: This study first reveals that, although CTLs with TCRs restricted to EBV antigens and therefore intrinsically low allo-specificity were used, CAR-independent lysis was observed in 24 hours co-culture with BCP-ALL cells. These results underline the need for cautious interpretation of cytotoxicity test results in an allogeneic setting. Secondly, these preliminary data suggest that an anti-HER2 CAR T-cell approach could be promising for BCP-ALL even with weak antigen expression. Anti-EBV CTLs have already been used as a platform for CAR-engineered T-cells (Rossig et al. 2017). Should the cytotoxic activity of allogeneic EBV-CTL be proven to be specifically directed against BCP-ALL, the synergy between TCR and anti-HER2-CAR might be beneficial, and adoptive transfer of anti-HER2 CAR EBV CTLs could be a promising therapeutic option for patients with HER2+ BCP-ALL. * This research is funded by the non-profit organization Etoile de Martin/SFCE.

Genetic Subtype Driven Changes in Bone Marrow Stromal Cells of Children with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction We previously showed that B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells hijack the normal bone marrow microenvironment to create a leukemic niche which facilitates blast cell survival and promotes drug resistance [1-3]. Bone marrow-derived mesenchymal stromal cells (MSCs) are a major component of the bone marrow microenvironment . Ex vivo,these cells provide a survival benefit to co-cultured leukemic cells and induce resistance to chemotherapeutic drugs [1, 2]. We previously showed that leukemic cells re-sensitize to chemotherapeutics ex vivo when the interaction with MSCs is disrupted [2]. Results We here report that patients' BCP-ALL cells induce an Interferon (IFN)-related gene signature in patients'-derived MSCs; a.o. IFI6, MX1, and OAS3 were 4.3 to 7.7-fold upregulated in MSCs flow-sorted after co-culture with BCP-ALL cells compared to the same MSCs flow-sorted after mono-culture . BCP-ALL samples (n=15) with an ETV6-RUNX1 translocation (5 out of 8 samples) were the most potent inducers of the IFN-related gene expression signature in MSCs compared to B-other BCP-ALL (1 out of 6 samples), with IFI6 3.0-fold (p=0.03), MX1 2.6-fold(p=0.05) and OAS3 2.1-fold (p=0.04) higher in MSCs after co-culture with ETV6-RUNX1 BCP-ALL cells. Similarly, an ETV6-RUNX1cell line (REH) also induced this IFN-response more strongly in MSCs compared to cell lines representing other BCP-ALL subtypes (SUPB15, MUTZ5, RCH-ACV and NALM6). Addition of specific inhibitors to these ex vivo BCP-ALL and MSCs cultures, revealed that the ETV6-RUNX1 induced IFN signature was dependent on IFNα/β signaling, whereas no such effect was seen for inhibitors of IFNγ signaling. Additionally, inhibition of direct contact between BCP-ALL cells and MSCs also negatively affected the expression of some IFN responsive genes in MSCs ( CXCL10 3.3-fold, p=0.02, IFI44L 1.57-fold, p=0.01 and IFITM1 1.5-fold, p=0.005) while other genes remained unaltered, suggesting that the transcriptional changes in MSCs are driven by close contact with BCP-ALL cells and not by soluble factors released by these cells or those being part of the culture medium. Normal immune cells including various populations of T-cells, monocytes, NK cells and dendritic cells did not alter the expression levels of the IFN-related genes in MSCs. We also observed that, while MSCs provide a survival benefit to (ETV6-RUNX1) BCP-ALL cells and induce resistance to chemotherapeutic drugs (prednisolone, daunorubicin, L-asparaginase) in these leukemic cells, this was not counteracted by inhibition of IFNα/β signaling. Conclusion Our data show that leukemic cells, more specifically ETV6-RUNX1 positive BCP-ALL cells, induce an IFNα/β signature in MSCs. Activation of this IFNα/β pathway in MSCs does not cause resistance of BCP-ALL cells to chemotherapeutic drugs and hence the IFNα/β activation may serve a different role, e.g., affecting the migration (recruitment and/or repelling) of specific immune cells to the leukemic bone marrow niche. Our findings warrant further studies into the role of the BCP-ALL-induced IFNα/β response in chemotaxis of healthy immune cells to create a beneficial microenvironment for BCP-ALL cells. This knowledge might point to alternative, maybe less intensive ways to eradicate leukemic cells.

RISK FACTORS AND EARLY SIGNS OF CRITICAL CONDITIONS IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA ADMITTED TO THE INTENSIVE CARE UNIT

Relevance: Acute lymphoblastic leukemia (ALL) is the most common cancer among children, accounting for nearly a quarter of all childhood cancers.&#x0D; The study aimed to determine the risk factors and signs of critical conditions in children with acute lymphoblastic leukemia admitted to an intensive care unit (ICU).&#x0D; Methods: The approach used was a systematic review. Data was collected from sources published in 2019-2023. Four cohort studies, four retrospective analyses, two literature reviews, one case-control study, and one case study were included in this systematic review.&#x0D; Results: The prognosis in pediatric ALL depends on the initial number of blast cells in the peripheral blood. Patients with B-cell precursor acute lymphoblastic leukemia (BCP ALL) and low blast cell numbers survived better than patients with T-cell acute lymphoblastic leukemia (T-ALL) and low cell count. IL1B and NLRP1 genetic polymorphisms enhanced ALL risk and reduced infectious comorbidity. However, these gene polymorphisms must be confirmed in juvenile leukemia. KRAS, FLT3, NRAS, PTPN11, KMT2D, PTEN, and NOTCH1 gene mutations affected pediatric ALL patient features and treatment results. These mutations demonstrate the relevance of genetic profiling in risk classification and tailored management. Gene variations and availability of effective medication contributed. Pediatric BCP-ALL patients with the PAX5P80R mutation had worse 5-year overall survival, higher white blood cell counts, male preponderance, and more genetic abnormalities. Pediatric BCP-ALL focused on genetic analysis and risk stratification. Children of African American and European American ancestry showed varied incidence, recurrence, and outcome rates for ALL. African American children exhibited lower incidence but greater recurrence rates and poorer prognosis than European American children.&#x0D; Conclusion: Risk factors for these patients’ admission to ICU include comorbidities, infectious diseases, hypoxia, and hemodynamic instability, as well as age and baseline white blood cell count at diagnosis.

Functional Characterization of Transforming Growth Factor-β Signaling in Dasatinib Resistance and Pre-BCR+ Acute Lymphoblastic Leukemia.

The multi-kinase inhibitor dasatinib has been implicated to be effective in pre-B-cell receptor (pre-BCR)-positive acute lymphoblastic leukemia (ALL) expressing the E2A-PBX1 fusion oncoprotein. The TGFβ signaling pathway is involved in a wide variety of cellular processes, including embryonic development and cell homeostasis, and it can have dual roles in cancer: suppressing tumor growth at early stages and mediating tumor progression at later stages. In this study, we identified the upregulation of the TGFβ signaling pathway in our previously generated human dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells using global transcriptomic analysis. We confirm the upregulation of the TGFβ pathway member SMAD3 at the transcriptional and translational levels in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Hence, dasatinib blocks, at least partially, TGFβ-induced SMAD3 phosphorylation in several B-cell precursor (BCP) ALL cell lines as well as in dasatinib-resistant pre-BCR+/E2A-PBX1+ ALL cells. Activation of the TGFβ signaling pathway by TGF-β1 leads to growth inhibition by cell cycle arrest at the G0/G1 stage, increase in apoptosis and transcriptional changes of SMAD-targeted genes, e.g. c-MYC downregulation, in pre-BCR+/E2A-PBX1+ ALL cells. These results provide a better understanding about the role that the TGFβ signaling pathway plays in leukemogenesis of BCP-ALL as well as in secondary drug resistance to dasatinib.

A new hope for early T cell precursor acute lymphoblastic leukemia therapy based on STAT5+ leukemic stem cell targeting.

Leukemia stem cells are known to drive tumor progression, drug resistance, microenvironmental shift, and relapse, which would make them a perfect therapeutic target. However, their phenotypic and functional similarity to their normal counterparts leaves limited road maps for their selective elimination. Tremblay et al. recently unraveled the fundamental role of overactivated pSTAT5 as a functional marker of early T cell precursor acute lymphoblastic leukemia stem cells driving leukemic progression and highlighted its potential use as a therapeutic target to prevent fatal outcomes.

Results of the compassionate program of inotuzumab ozogamicin for adult patients with relapsed or refractory acute lymphoblastic leukemia in Spain.

The prognosis of relapsed B cell precursor acute lymphoblastic leukemia (B-ALL) is poor and few patients can be successfully rescued with conventional therapies. Inotuzumab ozogamicin (IO), an antibody against the CD22 antigen linked to calicheamicin, has been approved as a rescue treatment in relapsed/refractory (R/R) B-ALL. This was an observational, retrospective, multicenter study of adult patients included in the Spanish program of compassionate use of IO in centers from the PETHEMA group (Programa Español de Tratamientos en Hematología). Thirty-four patients with a median age of 43 years (range, 19-73) were included. Twenty patients (59%) were refractory to the last treatment, IO treatment was given as ≥3rd salvage treatment in 25 patients (73%) and 20 patients (59%) received allogeneic hematopoietic stem cell transplantation before IO treatment. After a median of 2 cycles of IO, 64% of patients achieved complete response (CR)/complete response with incomplete recovery. The median response duration, progression-free survival and overall survival (OS) were 4.7 (95%CI, 2.4-7.0 months), 3.5 (95%CI, 1.0-5.0 months) and 4 months (95%CI, 1.9-6.1 months) respectively, with better OS for patients with relapsed B-ALL versus refractory disease (10.4 vs. 2.5 months, respectively) (p = .01). There was a trend for better OS for patients with first CR duration >12 months (7.2 months [95%CI, 3.2-11.2] vs. 3 months [95% CI, 1.8-4.2] respectively) (p = .054). There was no sinusoidal obstruction syndrome (SOS) event during IO treatment, but three patients (9%) developed grade 3-4 SOS during alloHSCT after IO treatment. Our study showed slightly inferior outcomes of the pivotal trial probably due to poorer risk factors and late onset of IO therapy of recruited patients. Our results support early use of IO in relapsed/refractory ALL patients.

Leukaemic cells expressing ETV6::FRK are sensitive to dasatinib in vivo.

ETV6::Fyn-related kinase (FRK), which is a Src family tyrosine-kinase-related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B-ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK-STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p<0.01). Our data support the potential of dasatinib as a therapeutic option for patients with B-ALL harboring FRK rearrangements.

Role of Cytogenetics and FISH in Laboratory Workup of B Cell Precursor Acute Lymphoblastic Leukemia

AbstractModern therapeutic protocols in acute leukemias risk stratify disease based on genetic characterization of the neoplastic cells and their response to treatment. Genetic characterization is routinely performed by cytogenetic testing of leukemic cells and is a standard component of modern risk-adapted therapy in acute lymphoblastic leukemia (ALL). High-throughput technologies like RNA sequencing have identified multiple novel subtypes in recent years. The cytogenetic strategy using GTG and fluorescent in-situ hybridization (FISH) has to be adapted to identify not only the primary principal chromosomal abnormalities but also the novel subtypes. In the review, we describe a systematic comprehensive cytogenetic strategy that integrates information from immunophenotyping, flow-based DNA ploidy, and karyotyping complemented by targeted FISH studies to identify more than 70% of genetic abnormalities described in B cell precursor ALL. The simplified strategy includes a four-probe FISH and flow ploidy strategy, ± karyotyping that identifies high risk (KMT2A, BCR::ABL1, hypodiploidy, iAMP21) and standard risk (ETV6::RUNX1 and high hyperdiploid) cytogenetic groups. The extended FISH panel includes probes targeting MEF2D, ZNF384, and CRLF2 rearrangements that are used intuitively on integrating the immunophenotyping features that characterize these entities. The strategy also includes a systematic approach to identify masked hypodiploidy integrating targeted FISH analysis directed toward identifying monosomies of chromosomes 7, 15, and 17 and flow cytometry-based DNA ploidy analysis. The recently described PH-like ALL is characterized by ABL class fusions and rearrangements of CRLF2 and JAK2 genes. FISH analysis using break-apart probes can be used to identify these aberrations. The cytogenetic approach also includes FISH analysis to identify intragenic and whole gene deletions of the IKZF1 genes that identify a subset of patients associated with high risk of treatment failure.

Comparison of flowcytometry-based scoring system for the diagnosis of early T precursor-acute lymphoblastic leukemia.

Early T cell precursor-acute lymphoblastic leukemia (ETP-ALL) is a hematolymphoid malignancy where the blasts demonstrate T cell differentiation markers along with stem cell and myeloid antigen expression. The differential diagnosis of ETP-ALL from non-ETP ALL and mixed phenotype acute leukemia is often challenging due to its overlapping immunophenotypic picture with co-expression of myeloid antigens. In this study, we endeavored to describe the immune-phenotype profile of ETP-ALL in our patients and compared the utility of four different scoring systems for better discrimination of these entities. This retrospective analysis included 31 ETP-ALL out of 860 acute leukemia cases consecutively diagnosed at the two tertiary care centers. Flowcytometry-based immunophenotype was reviewed for all the cases, and the utility of four flow-based objective scorings was assessed for the diagnosis of ETP-ALL. Receiver operating curves were drawn to compare the different flow-based scoring systems. The prevalence of ETP-ALL was 40% (n=31/77 T-ALL) in our study group, comprised mainly of adults with a median age of 20 years. The five-marker scoring system had the maximum area under the curve, followed by the seven-marker scoring system. A cut-off of ≥2.5 was more specific (sensitivity: 91%; specificity: 100%), while a score of ≥1.5 was more sensitive but slightly less specific (sensitivity: 94%, specificity: 96%). The WHO criteria for the diagnosis of ETP-ALL should be followed across all laboratories to avoid confusion and for better treatment stratification. Flow-based scoring systems can be objectively employed for better detection of cases.

The role of microenvironment in the initiation and evolution of B-cell precursor acute lymphoblastic leukemia.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a malignant disorder of immature B lineage immune progenitors and is the commonest cancer in children. Despite treatment advances it remains a leading cause of death in childhood and response rates in adults remain poor. A preleukemic state predisposing children to BCP-ALL frequently arises in utero, with an incidence far higher than that of transformed leukemia, offering the potential for early intervention to prevent disease. Understanding the natural history of this disease requires an appreciation of how cell-extrinsic pressures, including microenvironment, immune surveillance and chemotherapy direct cell-intrinsic genetic and epigenetic evolution. In this review, we outline how microenvironmental factors interact with BCP-ALL at different stages of tumorigenesis and highlight emerging therapeutic avenues.

Blinatumomab therapy for B cell acute lymphoblastic leukemia accompanied by persistent or relapsed low-level MRD prior to hematopoietic stem cell transplantation in Chinese children: a case series

BackgroundBlinatumomab could be successfully used to reduce minimal residual disease (MRD) prior to hematopoietic stem cell transplantation (HSCT) in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), but sound evidence is lacking in China.Case presentationThis retrospective study assessed the application of blinatumomab in B-ALL accompanied by persistent or relapsed low-level MRD before HSCT from April 2019 to July 2021. Two cases (Cases 1 and 2) initially achieved remission with MRD < 0.01% upon conventional therapy but had MRD relapse with MRD ≥ 0.01% but < 1% during maintenance treatment. Case 3 had no response to routine treatment, with high MRD (9.88% and 1.23% at days 19 and 46, respectively). Nevertheless, all patients had undetectable MRD. Cases 2 and 3 had undetectable fusion gene following blinatumomab therapy. By bone marrow monitoring (bone marrow morphology, bone marrow MRD and fusion gene) post-HSCT, the patients were persistently negative until May 15, 2022. No patient had serious adverse events before or during blinatumomab treatment.ConclusionsBlinatumomab therapy showed a good performance for three pediatric cases with detectable but low MRD before HSCT in China. However, further prospective studies with large sample sizes are still needed for further clarification.

Ph-like and IKZF1plus Features in Children with Down Syndrome and B Cell Precursor Acute Lymphoblastic Leukemia

Ph-like and IKZF1plus Features in Children with Down Syndrome and B Cell Precursor Acute Lymphoblastic Leukemia

A Human iPSC Model Reveals Specific Upregulation of Linker Histone H1-0 in ETV6::RUNX1+ Preleukemia and B Cell Precursor Acute Lymphoblastic Leukemia

The ETV6::RUNX1 translocation is the most common chromosomal rearrangement associated with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL). As previously shown, ETV6::RUNX1 leads to a distinct clinically silent preleukemic state in utero that manifests in impaired early B cell differentiation. Acquisition of secondary oncogenic mutations gives rise to overt leukemia in 0.2% of children carrying ETV6::RUNX1. In-depth characterization of ETV6::RUNX1+ preleukemia is essential to gain an understanding of leukemic transformation and develop preventive strategies. Here, we identify dysregulation of the epigenetic regulator H1-0 in ETV6::RUNX1+ preleukemia and BCP-ALL. Utilizing CRISPR/Cas9 genome editing, we generated monoclonal ETV6::RUNX1+ human induced pluripotent stem cell (hiPSC) lines with stable ETV6::RUNX1 expression driven by the endogenous ETV6 promoter. To assess the preleukemic setting mediated by ETV6::RUNX1, we performed RNA sequencing (RNA-seq) analysis of ETV6::RUNX1+ and matching wild-type (WT) hiPSCs of two donors. We identified 20 differentially expressed genes in ETV6::RUNX1+ hiPSCs of both donors compared to the respective WT hiPSCs (up: 15, down: 5, absolute fold change ≥ 2, p ≤ 0.05). Interestingly, our RNA-seq analysis revealed consistent upregulation of H1-0 (mean: 2.43-fold), a linker histone involved in chromatin remodeling, cell proliferation and differentiation. Elevated H1-0 levels in the ETV6::RUNX1+ hiPSC lines were confirmed by quantitative real-time PCR and immunoblot (Figure 1A). To determine H1-0 levels in ETV6::RUNX1+ B cell precursors, we analyzed published gene expression microarray and RNA-seq data sets. Both ETV6::RUNX1+ CD34+ cells (n = 10) derived from umbilical cord blood (Polak et al. Haematologica 2019) and ETV6::RUNX1+ CD34+CD19+ cells (n = 4) generated by hematopoietic differentiation of hiPSCs (Böiers et al. Developmental Cell 2018) showed significant upregulation of H1-0 compared to the respective control cells lacking ETV6::RUNX1 (unpaired t-test, p = 0.0087 and p = 0.0076, respectively). Additionally, we differentiated ETV6::RUNX1+ and WT hiPSCs into CD34+CD45+ hematopoietic progenitor cells and performed single-cell RNA-seq analysis. We identified coexpression of H1-0 with cyclin-dependent kinase inhibitor genes CDKN1A and CDKN1B, indicating a slow or non-cycling phenotype of H1-0+ cells. Next, we examined H1-0 mRNA levels in gene expression data sets of pediatric and adult leukemia patients (St. Jude PeCan database (n = 876), Jerchel et al. Haematologica 2018 (n = 654), MILE study (n = 1299)). H1-0 expression was found to be significantly elevated in ETV6::RUNX1+ BCP-ALL patient samples compared to other leukemia entities, especially TCF3::PBX1+ ALL (ordinary one-way ANOVA, p ≤ 0.0001), KMT2A rearranged ALL (p ≤ 0.0001) and chronic lymphoblastic leukemia (CLL, p ≤ 0.0001). Strikingly, H1-0 expression was 22.37-fold lower in CLL compared to ETV6::RUNX1+ BCP-ALL (Figure 1B). This observation prompted us to investigate H1-0 expression in B cell developmental stages using published data sets. Interestingly, we found that H1-0 levels decrease during early B cell development in both normal bone marrow (Black et al. Nucleic Acids Research 2018) and umbilical cord blood-derived cells (Novershtern et al. Cell 2011). Additionally, analysis of single-cell RNA-seq data of early B cell precursors derived from healthy bone marrow (Mehtonen et al. Genome Medicine 2020) and fetal liver (Developmental Cell Atlas Newcastle University) revealed that relative numbers of H1-0 expressing cells per differentiation state decreased along the pseudotime B cell development trajectory. Taken together, our study identifies H1-0 upregulation as a specific alteration present already at the ETV6::RUNX1+ preleukemic state. High H1-0 expression was associated with slower proliferation and increased stemness. This phenotype might explain the persistence of in utero generated preleukemic ETV6::RUNX1+ clones residing in the bone marrow of approximately 5% of healthy children for years. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IKZF1 Deletions Induce Resistance to AraC in BCP-ALL Reversible By CDK8/19 Inhibition

Background Deletions and mutations affecting lymphoid transcription factor IKZF1 occur in about 10-15% of pediatric patients and up to 50% of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) and predict a poor outcome. We have shown previously that loss of IKZF1 function compromises the therapeutic efficacy of glucocorticoids (GCs). Here, we investigated whether loss of IKZF1 function also affects therapy response to other chemotherapeutic agents used in the treatment of ALL. Aim To develop tailored treatment protocols for IKZF1 deficient leukemia Results We used CRISPR-Cas9 to target the IKZF1 locus in SEM pro-B ALL cells and evaluated the response to standard chemotherapy used in the treatment of ALL. We observed that IKZF1-/- SEM cells show a markedly reduced response to the pyrimidine analogue cytarabine (AraC) relative to IKZF1 wild type cells, with an 8-fold increase in IC50. The same drug resistance profile was observed upon shRNA-mediated knockdown of IKZF1 as well as in B-cells isolated from 8 to 14 weeks-old Ikzf1+/- mice. Using ex vivo cultures of patient derived xenografts (PDX), we observed that IKZF1 deficient leukemic cells were significantly more resistant to AraC relative to cells wild type for IKZF1 (3-fold increase in area under the curve (AUC) (Figure A). In addition, we targeted the IKZF1 protein for proteasomal degradation using the thalomide Iberdomide in PDX cells wildtype for IKZF1, showing that this drug-induced IKZF1 loss leads to AraC resistance in 7 out of 8 PDX samples. To provide in-patient evidence for AraC resistance, we compared the reduction in tumor load between patients with leukemias wildtype for IKZF1 with those carrying IKZF1 deficiencies after treatment with an AraC containing treatment block (cumulative dose of 1200 mg/m2 AraC, 6-mercaptopurine 60 mg/m2/day p.o., for 28 days and 2 doses of 1000 mg/m2 cyclophosphamide). In the Dutch ALL10/11 protocol, this treatment block is flanked by minimal residual disease (MRD) measurements. We selected all patients with a high tumor load (MRD of 10-2) before the start of the AraC containing block and calculated the reduction in leukemia burden by comparing MRD levels before and after treatment with the AraC containing interval. This showed that the response to AraC treatment was attenuated in patients with IKZF1 deleted ALL (n=25) compared to patients WT for IKZF1 (n=35) (**p=0.0065). Because IKZF1 deficient cells did not show resistance to other compounds used in this treatment block, we attribute the reduced treatment response to increased AraC resistance. To further confirm the involvement of IKZF1 loss in AraC resistance, we performed a single mouse trial in which we compared AraC treatment response in mice transplanted with IKZF1 wildtype leukemias (n=5) with IKZF1 deficient leukemias (n=7), confirming that the latter respond more poorly to treatment. To study whether resistance in response to IKZF1 loss can be reversed and obtain more insights into the biology behind the observed AraC resistance, we performed CRISPR/Cas9 screens to identify drug targets that can restore response to AraC therapy. Briefly, IKZF1 deficient cells were transduced with a library of gRNAs targeting subset of genes (kinases and genes involved in cellular metabolism). Transduced cells were treated with a sublethal dose of AraC and the relative abundance of each gRNA before and after treatment was calculated. In addition to known modifiers of the AraC therapy response such as deoxycytidine kinase, we also identified novel targets that may be targeted to sensitize cells to cytarabine treatment. Guide RNAs targeting the mediator kinases CDK8/19 were selectively and strongly depleted during treatment, suggesting that these proteins modify response to cytarabine treatment. Indeed, small-molecule inhibitors targeting CDK8/19 kinase activity synergized with cytarabine induced killing of leukemic cells (Figure B). Conclusion Together, our results demonstrate that loss of IKZF1 function confers resistance to AraC in BCP-ALL. Moreover, this resistance can be overcome by use of small molecule inhibitors such as those targeting CDK8/19. We expect that these studies will aid in the rational design of combination therapies that will further improve treatment response in these high-risk leukemias. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Multi-Cohort Gene Expression Classifier (ALLCatchR) Identifies B-Precursor ALL Subtypes and Their Developmental Trajectories across Age Groups

The molecular landscape of B cell precursor acute lymphoblastic leukemia (BCP-ALL) describes >20 disease subtypes defined by initiating genomic alterations and corresponding gene expression signatures. Some subtypes represent phenocopies with shared gene expression signatures but distinct or absent genomic drivers. The majority of BCP-ALL subtypes have been acknowledged as diagnostic entities by the currently revised classifications of hematologic malignancies (WHO-HAEM5 / International Consensus Classification). Thus, unified diagnostic approaches are needed to systematically identify subtypes in individual BCP-ALL cases in clinical routine. Gene expression signatures represent the shared functional equivalent of heterogeneous genomic driver alterations. Therefore, we established a gene expression-based classifier to identify BCP-ALL subtypes and related clinical and biological phenotypes. We analyzed a total of n=3477 BCP-ALL gene expression profiles obtained from transcriptome sequencing of adult (n=1253) and pediatric patients (n=2224) including publicly available (n=2074; Gu Z et al. Nat. Genet. 2019; Schmidt B. et al. Blood Adv. 2022) and own data sets (n=1403) from adult and pediatric European study groups (GMALL, AIEOP-BFM, CELL). Raw read counts from 6 cohorts representing heterogeneous library preps (i.e., stranded/unstranded; mRNA/total RNA), sequencing platforms/depths, and gene count quantifications were included. All samples had been previously allocated to molecular BCP-ALL subtypes based on integration of independent genomic profiling, gene fusion calling and gene expression. For classification, data with defined subgroup allocation were randomly split into training (n=1831; 70%) and testing sets (n=785; 30%) and two hold-out studies (n=305). To define gene expression signatures of 21 BCP-ALL subtypes, we used training set samples and extracted specific gene sets employing different feature selection algorithms as well as ranking of genes in individual subtypes. Based on these definitions we established a BCP-ALL subtype classifier (ALLCatchR), which integrates machine learning models (support vector machines) and nearest-neighbors associations of single sample gene set enrichment analyses (Fig. A). ALLCatchR performed molecular subtype allocation for the testing set at high sensitivity (0.934±0.165) and specificity (0.997±0.005) on average across subtypes. A similar performance was achieved in the two hold-out cohorts despite completely independent data structures which have not been shown to the classifier (sensitivity: 0.946±0.097 / specificity: 0.996±0.007). Overall accuracies of 0.952 and 0.92 for testing and hold-out-data sets indicate that ALLCatchR reliably identifies molecular BCP-ALL subtypes in mixed adult / pediatric cohorts based on gene expression profiles alone (Fig. A, below). Among n=44/1090 (4%) samples of the combined hold-out/testing set with ambiguous subtype allocation (deemed 'unclassified'), ALLCatchR provides candidate subtype allocations (sensitivity: 0.671±0.393 / specificity: 0.978±0.036) for confirmation based on genomic driver alterations. In addition, we modified our classifier to predict baseline variables such as patient's sex (accuracy: 0.991), immunophenotype (accuracy: 0.935) or blast count (R=0.633). To define the corresponding B cell differentiation stage of origin, we compared BCP-ALL subtypes to differentiation stage-specific gene sets extracted from 7 FACS-sorted lymphopoietic subsets from healthy bone marrow donors (n=4). We observed defined BCP-ALL subtype-specific enrichment patterns which can be grouped along 4 major differentiation stages (Pro-B to Pre-B-II-Large, Fig. 1B) with a high degree of similarity in independent adult and pediatric cohorts. Thus, B cell developmental trajectories underlying BCP-ALL subtypes are dissected at high resolution by gene expression profiling and differentiation stages may favor the selection of specific leukemogenic drivers. ALLCatchR is a gene expression classifier for 21 established BCP-ALL molecular subtypes, which performs at independently validated cohorts with an accuracy of >90% and allows imputation of clinical baseline variables as well as underlying B cell developmental trajectories. It provides a reliable basis for systematic diagnostic approaches in pediatric and adult BCP-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal