B-cell Precursor Acute Lymphoblastic Leukemia Research Articles

An Attempt to Determine Hematological Malignancies in Selected Patients: A Cytogenetical Approach

Diagnosis and treatment of hematological malignancies, a diverse category of tumors that begin in the blood and bone marrow, are extremely difficult. Our knowledge of these conditions has been revolutionized by the development of cytogenetics, the study of chromosomes and genetic abnormalities. A total of 180 patients with benign and malignant hematological malignancy had peripheral bone marrow or blood samples obtained using the standard G-banding procedure. Fifty-one percent of patients had normal karyotypes. In addition, thirty individuals (15%) had a complicated karyotype whereas eighty-five percent (85%) had a normal one. Three-hundred and three percent of patients were diagnosed with Pre-B Acute Lymphoblastic Leukemia (Pre-B ALL), followed by twenty percent with Chronic Myelogenous Leukemia (CML), and fourteen percent with Acute Lymphocytic Leukemia.

Multimodal classification of molecular subtypes in pediatric acute lymphoblastic leukemia

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

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.

Clinical and prognostic significance of CD27 and CD44 expression patterns in Egyptian pediatric patients with B-precursor acute lymphoblastic leukemia

IntroductionThe expression pattern of CD27 and CD44 was found to correlate with the differentiation stages of B cell precursors, which were known to be involved in a variety of immunological responses. Aim of the studyThis study aimed to determine the biological significance of CD27 and CD44 expression in patients with B-precursor acute lymphoblastic leukemia (B-ALL), as well as their association with standard prognostic factors and therapeutic response. Patients and methodsThis case-control study included 60 pediatric patients newly diagnosed with B-ALL and 30 pediatric controls. The patient CD27 and CD44 levels were measured using the Beckman Coulter Navios Flow Cytometer. ResultsMost malignant cells (91.6 %) expressed CD44, but only 50 % of the patients had CD27 expressed. The positive CD 44 expression was associated with unfavorable prognostic markers, including a decrease

Functional relevance of circRNA aberrant expression in pediatric acute leukemia with KMT2A::AFF1 fusion

AbstractCircular RNAs (circRNAs) are emerging molecular players in leukemogenesis and promising therapeutic targets. In KMT2A::AFF1 (MLL::AF4)-rearranged leukemia, an aggressive disease compared with other pediatric B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), data about circRNAs are limited. Here, we disclose the circRNA landscape of infant patients with KMT2A::AFF1 translocated BCP-ALL showing dysregulated, mostly ectopically expressed, circRNAs in leukemia cells. Most of these circRNAs, apart from circHIPK3 and circZNF609, previously associated with oncogenic behavior in ALL, are still uncharacterized. An in vitro loss-of-function screening identified an oncogenic role of circFKBP5, circKLHL2, circNR3C1, and circPAN3 in KMT2A::AFF1 ALL, whose silencing affected cell proliferation and apoptosis. Further study in an extended cohort disclosed a significantly correlated expression of these oncogenic circRNAs and their putative involvement in common regulatory networks. Moreover, it showed that circAFF1 upregulation occurs in a subset of cases with HOXA KMT2A::AFF1 ALL. Collectively, functional analyses and patient data reveal oncogenic circRNA upregulation as a relevant mechanism that sustains the malignant cell phenotype in KMT2A::AFF1 ALL.

Characterizing Morphometric and Nanomechanical Malignant Cell Features in a Rare Paediatric γδ T-acute Lymphoblastic Leukaemia: Insights from a Single Case Study Using Atomic Force Microscopy

The most common cancer in paediatric age is acute lymphoblastic leukaemia (ALL) which accounts for nearly a quarter of all cases of paediatric cancer. ALL cases are classified as B-cell or T-cell precursor ALL, based on their immunophenotypical features. Patients with T-cell ALL (T-ALL) comprise 10–15% of all newly diagnosed cases and are more prevalent in boys and in older age compared to the overall incidence peak age of ALL. In this case report, we present the morphometric and nanomechanical features of T-lymphoblasts derived from an 11-month-old infant with a rare subtype of γδ T-ALL with aggressive biological behavior. To investigate these features, we employed atomic force microscopy (AFM) and compared the blast cell deviations found in their nanostructure and elasticity to those of normal lymphocytes from a healthy child. The malignant T-lymphoblasts exhibited reduced roughness and Young’s modulus values. This single case analysis demonstrates the potential of the AFM method to provide additional information regarding the characteristics of malignant cells and suggests its potential as a complementary approach for distinguishing neoplastic cells from normal cells. The application of AFM could potentially facilitate the introduction of in vitro tests to determine the efficacy of anti-leukemic treatments.

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.

Constitutional and Somatic Variants in ARID5B in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the high hyperdiploid (HeH) subtype accounting for approximately 25% of B-cell precursor (BCP) ALL cases. It has been shown that germline variants in the ARID5B gene in chromosome band 10q21.2 are associated with increased risk of BCP ALL, in particular HeH ALL, and that the risk alleles result in lower expression of ARID5B in hematopoietic cells. ARID5B codes for a protein involved in regulating gene expression and chromatin remodeling. We have previously reported somatic deletions in the ARID5B locus in two cases of HeH ALL, but the overall frequency of acquired copy number changes and rearrangements involving ARID5B in BCP ALL remains unknown. Here, we have investigated constitutional and somatic ARID5B variants in pediatric BCP ALL, with a particular focus on HeH cases. METHODS Constitutional variants We studied four known risk single nucleotide polymorphisms (SNPs) in the ARID5B locus (rs7090445, rs7089424, rs7073837 and rs10740055) in HeH ALL cases heterozygous for the risk SNP and with trisomy 10. These were investigated in three different cohorts, including a total of 92 cases informative for rs7090445, 92 cases for rs7089424, 119 cases for rs7073837 and 66 cases for rs10740055. The genotype and relative allele frequencies were ascertained from SNP array or whole genome sequencing (WGS) data. One-sided binomial tests were applied to investigate whether the risk allele was more often in the duplicated chromosome than the non-risk allele. Somatic variants For somatic variants, we ascertained copy number status based on SNP array and/or WGS analysis in the ARID5B region in a total of 466 pediatric HeH ALL cases and structural rearrangements based on WGS in 77 cases. We also studied, using SNP array analysis, somatic copy number variants in a separate cohort consisting of 590 non-HeH BCP ALL cases. To compare the proportions of deletions in the HeH cohort and in the other genetic subtypes, we used Fisher's Exact two-sided test. RESULTS Constitutional variants All four risk SNPs showed a significantly higher proportion of risk allele duplication (one-sided binominal test); rs7090445 ( P=0.009), rs7089424 ( P=0.005), rs7073837 ( P=0.03) and rs10740055 ( P=0.04). This validates that there is a clonal selection for HeH blast cells with gain of the chromosome 10 homologue carrying the risk allele as opposed to gain of the homologue that carries the non-risk allele. Somatic variants Somatic deletions targeting ARID5B were found in 9/466 cases (1.9%) of HeH cases. The deletions covered different parts of ARID5B, with no minimally deleted region, suggesting that the functional outcome was downregulation of ARID5B expression. In the cohort with non-HeH BCP ALL (other genetic subtypes), 4/590 somatic deletions were found (0.68%). There was no statistically significant difference between the frequency of copy number aberrations targeting ARID5B between HeH and the other genetic subtypes of ALL (P=0.09). WGS analysis of the HeH cases revealed one translocation and one missense mutation in 2/77 cases (2.6%). The translocation involved the PAN3 and ARID5B genes and was also present in RNA sequencing data from this case. CONCLUSIONS We show that our previous finding that HeH ALL constitutionally heterozygous for ARID5B risk alleles and with an acquired trisomy 10 more commonly duplicates the chromosome 10 homologue carrying the risk allele holds true in a much larger cohort. Furthermore, somatic deletions involving ARID5B are recurrent in pediatric BCP ALL.

The 3D Chromatin Landscape of B-Cell Precursor Childhood Acute Lymphoblastic Leukemia

Whilst the molecular pathogenesis of childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) has been studied extensively, its 3D chromatin landscape remains poorly explored. Genome-wide chromosome conformation capture methods have provided the tools to investigate the different units of chromatin organization, such as transcriptionally active (A) and inactive (B) compartments, topologically associating domains (TADs), and fine-scale chromatin loops and enhancer-promoter interactions. The aim of this study is to elucidate the chromatin architecture and topological gene regulation in childhood BCP ALL. To date, 29 primary patient samples were included, comprising the high hyperdiploid (HeH) (n=11), ETV6:: RUNX1-positive (n=8), BCR:: ABL1-positive (n=2), TCF3:: PBX1-positive (n=2), DUX4-rearranged (n=2), intrachromosomal amplification of chromosome 21 (iAMP21) (n=1), KMT2A-rearranged (n=1), near-haploid (n=1) and near-triploid (n=1) genetic subtypes. Leukemic blast cells obtained at diagnosis were analyzed using Micro-C, a high-resolution variation of Hi-C (average number of total reads = 1.4 billion, highest resolution = 5 kb) combined with pair-end sequencing. Chromatin contact heatmaps were generated for each case using Juicer and Cooler. A/B compartments were identified using FanC at 500 kb resolution and visualized in the software IGV, while TAD calling was performed by Juicer, Domaincaller and Insulation Score. Differential chromatin interaction loop calling was made using Pareidolia and Mustache, and structural variant (SV) calling was carried out by EagleC. Preliminary principal component analysis of the 29 cases, based on the first two eigenvectors of the contact matrix (500 kb resolution), showed that HeH, ETV6:: RUNX1-positive, TCF3:: PBX1-positive and DUX4-rearranged cases each clustered based on their chromatin 3D organization. Furthermore, the A/B compartments of 11 HeH and 8 ETV6:: RUNX1-positive cases were analyzed at 500 kb resolution and compartment shifts among the two subtypes were annotated. A total of 390 shifts were detected, where activating shifts (from B to A compartment) happened more often in HeH (263 shifts) than in ETV6:: RUNX1-positive cases (127 shifts). Analysis of TAD boundary strength at 25 kb resolution revealed that HeH cases displayed significantly weaker boundaries compared to ETV6:: RUNX1-positive cases. TAD boundary strength showed no bias towards the frequently gained or non-gained chromosomes in HeH ALL. By merging individual heatmaps of all HeH and ETV6:: RUNX1-positive cases using Cooler, we created subtype-specific profiles and compared the intensity of chromatin interactions between the two genetic subtypes. Chromatin interaction intensity analysis was then combined with previously published RNA-sequencing data to identify transcriptional dysregulation events that could be associated with chromatin interaction changes. Preliminary results show that there was a chromatin loop missing close to the well-known leukemia-related gene IKZF1 in HeH compared to ETV6:: RUNX1-positive cases; this gene also showed lower expression in the RNA-sequencing data. FLT3 was associated with weakened chromatin interactions and down-regulated in ETV6:: RUNX1-positive cases compared to HeH, in agreement with its known high expression in HeH. Finally, we performed screening of SVs using EagleC and Micro-C heatmaps in HeH and ETV6:: RUNX1-positive samples. Out of the 19 included cases, previous whole-genome sequencing (WGS) data were available for 16. We detected 75 SVs, of which 50 were intrachromosomal rearrangements and 25 were translocations. Micro-C heatmaps allowed visual detection of SVs smaller than 1 Mb and permitted identification of the type of SVs. WGS detected 61% of the SVs found in the HeH samples with Micro-C and 51% of those in the ETV6:: RUNX1-positive cases. In summary, we present the first high-resolution genome-wide map of chromatin 3D organization in pediatric ALL. Our results indicate that different subtypes of childhood BCP ALL have distinct 3D chromatin landscapes and that abnormal chromatin architectures affect the regulation of leukemia-related genes.

CNS Invasion of TCF3::PBX1+ Leukemia Cells Requires Upregulation of AP-1 Signaling As Revealed By Brain Organoid Model

Introduction: Involvement of the central nervous system (CNS) remains a challenge in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Currently, the investigation of CNS leukemia mechanisms relies heavily on 2D cell culture and mouse models. However, given the differences between human and murine CNS in cellular identity and architecture, it becomes crucial to explore alternative models to study CNS leukemia. Moreover, novel targets for diagnosis and treatment of CNS ALL are critically needed. Methods: In this study, we established a pioneering 3D co-culture model that combines human induced pluripotent stem cell (iPSC)-derived cerebral organoids with BCP-ALL cells. We developed new methods to extract data from our invasion assays by enhancing the visualization of 3D organoid images, enabling us to accurately measure the invasion depth of leukemia cells compared to healthy controls within the organoids relative to their surface. To gain further insights on leukemia cells invading the organoids compared to those in the non-invaded fraction, we conducted RNA sequencing and immunofluorescence staining. Subsequently, we validated these results in a BCP-ALL in vivo mouse model and in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases within a cohort of 100 BCP-ALL patients. Results: Our experiments demonstrated robust and deep engraftment of TCF3::PBX1+ leukemia cell lines and patient-derived xenograft (PDX) cells into cerebral organoids (within a 14-day of co-culture). In contrast, the engraftment of healthy human CD34+ hematopoietic stem and progenitor cells (HSPCs) was limited ( p < 0.05) as compared to leukemia cells. Utilizing the co-culture model, we successfully validated the targeting of CNS leukemia-relevant pathways, such as CD79a/Igα or CXCR4-SDF1, via genetic knockdown or blocking experiments using inhibitor, respectively, which reduced the invasion of BCP-ALL cells into the organoids. Of note, RNA sequencing and immunofluorescence staining analysis revealed a significant upregulation of members of the AP-1 transcription factor complex, namely FOS, FOSB, and JUN, in the organoid-invading cells. Furthermore, we found an enrichment of AP-1 pathway genes in PDX cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX BCP-ALL cells (n = 5), thereby supporting the critical role of AP-1 signaling in CNS disease (Figure 1A). In line with these findings, we observed significantly higher levels of the AP-1 gene JUN in patients initially diagnosed with CNS-positive BCP-ALL compared to CNS-negative cases (mean JUN expression: 633.4 ± 78.51 in CNS-negative vs 1142 ± 296.5 in CNS-positive) within a cohort of 100 BCP-ALL patients (Figure 1B). Summary: In summary, we present ALL co-culture systems with iPSC-derived cerebral organoids as a promising complementary model to investigate CNS involvement in BCP-ALL including therapeutic targeting approaches, and identified the AP-1 pathway as a marker of CNS disease in TCF3::PBX1+ BCP-ALL.

CD38 Is a Functional Partner of CD9 in B-Cell Precursor Acute Lymphoblastic Leukemia

Background: We previously showed that CD9 was expressed in ~80% of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cases and its positivity was associated with an inferior relapse-free survival (Leung et al, Leukemia, 2020), suggesting its oncogenic role in this disease entity. Yet, the mechanisms underlying how CD9 affects leukemia progression remain completely unknown. As a prototypic member of the tetraspanin superfamily proteins, CD9 is able to organize with transmembrane or intracellular proteins to form the “tetraspanin web” to exert its modulatory functions. In BCP-ALL, though there has been a standalone study reporting 28 CD9-binding proteins in NALM-6 cells, the results are unlikely representative of the entire disease spectrum due to tumor heterogeneity. Here, we perform an in-depth proteomic study to dissect the CD9 web composition and downstream protein partners to drive disease progression in BCP-ALL. Methods: Immunoprecipitation coupled mass spectrometry (IP-MS) was performed to map the overall composition of CD9 web in five CD9 + BCP-ALL cell lines with distinct cytogenetic backgrounds, including 697 ( TCF3-PBX1+), BV-173 ( BCR-ABL1+), HAL-01 ( TCF3-HLF+), KASUMI-2 ( TCF3-PBX1+) and RS4;11 ( KMT2A-AFF1+). Overlapping protein partners were then validated by IP-Western blot (IP-WB) in 13 patient samples spanning major BCP-ALL subtypes. Shortlisted CD9 partners were further characterized by antibody blockade and CRISPR/Cas9 loss-of-function experiments to evaluate their impact on leukemia proliferation in vitro and leukemia progression in vivo, coupled with enzymatic and phenotypic assays to evaluate the direct impact of CD9 on partner functions. Results: A total of 278 CD9-binding proteins comprising 24 known and 254 novel partners were identified by IP-MS. Venn analysis revealed a marked heterogeneity of CD9-binding proteins across leukemia subtypes, with only 10 being intersected representing the “universal CD9 partners” in BCP-ALL. These proteins consisted of 5 established CD9 partners: ADAM10, CD19, CD38, CD81 and IGSF8 as well as 5 novel partners: ANXA11, HBG2, TMEM109, SLC44A1 and STXBP3. Two universal CD9 partners, namely CD38 and CD81, were consistently detected in all pediatric BCP-ALL samples. Neutralizing antibodies against CD9, CD38 or CD81 unanimously inhibited proliferation of 697 cells in vitro ( P<0.05). However, in the background of CD9 neutralization, CD81, but not CD38, offered an additional impact on leukemia suppression ( P<0.05). Likewise, CD38 knockout cells treated with CD9 antibody did not appear to inhibit cell proliferation in an additive manner. Concordantly, in the 697 xenografts, CD9 or CD38 antibodies alone prolonged animal survival by 2.1- and 1.6-fold, but combinational treatment did not produce the projected additive effect (2.5-fold only). In addition, CD9 neutralization significantly reduced the enzymatic activity of CD38 by 52.5% ( P<0.05) without altering its expression. Conclusions: This work presents the composition of the CD9 protein web in BCP-ALL, revealing homogenous and heterogeneous partners across the leukemia subtypes. We also provide definitive evidence showing that CD9 binds to and regulates CD38 activity to mediate leukemia progression, representing the first universal protein partner being identified to have a functional consequence in BCP-ALL, thereby providing a mechanistic basis to support the development of CD9-targeted therapies for this life-threatening malignancy.

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

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.

Mutually-Exclusive Pathways between IKZF1plus and RAS Mutations and TP53 double-Hit Alterations Lead Relapse in B-Other, Ph-like and Hyperdiploidy Genetic Groups in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a hematologic malignancy characterized by a high genetic heterogeneity and high relapse rate. Despite treatment advances have led to high cure rates in pediatric BCP-ALL (90%), improving survival in adults remains a challenge. Identification of genetic alterations driving relapse and both new prognostic and therapeutic biomarkers are important to improve survival in adults with BCP-ALL. Aim: To identify genetic alterations that lead to relapse in adult BCP-ALL patients (18-72 years) based on the tumor genetic landscape. Methods: Forty-five adult BCP-ALL patients (median age: 43 years; range 18-72 years) were studied at diagnosis and relapse by massive sequencing using a custom panel that allows detection of single nucleotide variants (SNVs) and insertions/deletions (INDELs) in 150 genes involved in BCP-ALL, in addition to copy number variations (CNVs), gene fusions and aneuploidy. Thirty-eight percent of patients belonged to the BCP-other ALL group, 16% were Philadelphia-positive (Ph+), 16% were classified as Philadelphia-like (Ph-like), 11% belonged to the high hyperdiploidy group, 8% had KMT2A rearrangements ( KMT2Ar), 4% TCF3::PBX1 fusion, 3% showed hypodiploidy, 2% low hyperdiploidy and 2% de novo MYC and BCL2 double-hit. Patients were treated according to the Spanish Adult-PETHEMA high-risk ALL protocols. Results: The most frequently deleted gene at diagnosis was IKZF1 (25/45, 56%). Among these cases, 72% (20/25) had IKZF1plus genetic profile, characterized by co-occurrence of IKZF1 deletions with at least one deletion in CDKN2A/B, PAX5 or PAR1, without ERG deletion. The IKZF1plus genetic profile was associated with specific genetic subgroups: Ph+ (5/7, 71%), Ph-like (5/7, 71%) and B-other (10/17, 59%). Moreover, it was significantly related with the presence of minimal/measurable residual disease (MRD) after induction (16/18, 89%) p < 0.001 and persistence at relapse in most patients (18/20, 90%; p < 0.001). Interestingly, among those 5 patients initially diagnosed with IKZF1 deletion but who did not meet the criteria for the IKZF1plus profile, 2 of them acquired the IKZF1plus profile at relapse. This suggests a possible cooperation of these genetic deletions contributing to therapy resistance and disease progression in these genetic subgroups. Eighty-six percent (6/7) of Ph+ patients who received imatinib treatment presented ABL1 mutations that were detected exclusively at relapse. Of these cases, 67% (4/6) were also associated with IKZF1plus at relapse (Figure 1A). Two mutually exclusive genetic patterns were identified at relapse in adult B-other and Ph-like patients. Thus, patients with IKZF1plus profile did not show RAS pathway mutations (59% and 57%, respectively), while patients with RAS pathway mutations lack in IKZF1plus profile (18% and 29%, respectively) (Figure 1B). Moreover age-related differences in the adult B-other ALL patient group were observed. The presence of mutations in RAS pathway was associated with younger age (<40 years) (3/10, 30% vs. 0/7, 0%), whereas older patients (≥40 years) predominated in the IKZF1plus profile (6/7, 86% vs. 3/10, 30%). TP53 alterations were detected at diagnosis and/or relapse in 11 patients (24%), most of which (9/11, 82%) had a heterozygous mutation plus a deletion or a homozygous mutation (double-hit). The high hyperdiploidy subgroup was enriched in TP53 mutations (4/5, 80%), most of which were already present at diagnosis (3/4, 75%). These findings indicate that genetic biomarkers usually linked to a favorable prognosis, such as high hyperdiploidy, can be associated with unfavorable genetic markers, leading to poorer outcomes in this group of patients (Figure 1C). Both the IKZF1plus profile and TP53 alterations were associated with poorer overall survival (OS) rates (P=0.034 and P=0.040, respectively) in adult BCP-ALL (Figure 2). Conclusion: Our results highlight the clinical importance of the TP53 alterations and the IKZF1plus profile as prognostic factors in adult BCP-ALL, suggesting they might help guiding more personalized therapeutic strategies to improve outcomes in this group of BCP-ALL patients. Grants: SACYL(GRS2386/A/21,GRS2385/A/21,GRS2527/A/22,GRS2506/A/22), JCyL SA118P20, FMM21/002 AP176752021, Pfizer 69383919.

Outcome of Children with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) with Hypodiploidy or BCR-ABL1 Fusion Given Allogeneic Hematopoietic Stem Cell Transplantation (HSCT): Results from the Prospective Forum Study

Introduction: Hypodiploid karyotype (HYPO) and bcr-abl1 fusion (BCRABL) define high-risk BCP-ALL subtypes, with historically worse outcomes. Many front-line treatment protocols recommend allogeneic HSCT for these patients (pts), either in first complete remission (CR1) irrespective of response (adolescents/young adults with HYPO), if minimal residual disease (MRD) signals persist at specific time points (younger pts with HYPO and all pts with BCRABL), or following leukemic relapse. We have assessed the outcomes of BCP-ALL pts aged 4 to 18 years (y) with either HYPO or BCRABL who participated in the phase III ALL SCT PED FORUM study (EudraCT: 2012-003032-22) and underwent HSCT from either a matched sibling (MSD) or matched unrelated donor (MUD). Patients and Methods: Between 2014 and January 2023, 741 evaluable BCP-ALL pts were enrolled in FORUM (Table 1). HYPO was present in 61 pts, BCRABL in 82 pts, and neither lesion in 598 pts (NEITHER). 43/61 (70%), 40/82 (49%) and 215/598 (36%) pts with HYPO, BCRABL and NEITHER were in CR1, respectively (p= n.s.). Prior to HSCT, 82 of 101 HYPO/BCRABL pts with available MRD data were MRD negative by PCR and/or flowcytometry (<10-4). Total body irradiation (TBI) + VP16 was administered to 42 and 50 pts in the HYPO and BCRABL groups, respectively, while 18 HYPO and 30 BCRABL pts received non-TBI conditioning (busulfan or treosulfan + fludarabine and thiotepa, as per study protocol). Randomization determined allocation to TBI/non-TBI treatment arms until randomization closure in 03/2019, while treatment in some pts was dictated by lack of access to TBI, or contraindication to TBI in individual pts. An MSD was available in 14 (23%) and 24 (29%) HYPO and BCRABL pts, respectively. Notably, 24 out of 143 pts (17%) with HYPO/BCRABL received immunotherapy before HSCT (blinatumomab, inotuzumab ozogamicin, and/or CAR-T cells). Post-HSCT, 14 out of 82 BCRABL pts (17%) received a tyrosine kinase inhibitor (TKI), either prophylactically or pre-emptively. Results: With a median follow-up of three years post-HSCT, the 5-y probabilities of overall survival (OS) for pts with HYPO, BCRABL and NEITHER were 0.79±0.06, 0.83±0.05, and 0.74±0.02 (p=n.s.), while those of event-free survival (EFS) were 0.73±0.06, 0.65±0.07, and 0.61±0.02, respectively (p=n.s.). The 5-y cumulative incidence of relapse (CIR) for HYPO/BCRABL and NEITHER pts was 0.21±0.04 and 0.29±0.02 (p=0.036), and 5-y non-relapse mortality (NRM) was nearly identical for these groups (0.07±0.2 and 0.08±0.1). Among pts who received TBI, 5-y OS and EFS for HYPO, BCRABL, and NEITHER groups were 0.76±0.08 and 0.70±0.08, 0.89±0.05 and 0.70±0.10, and 0.80±0.03 and 0.71±0.03, respectively (p=n.s., Figure 1), while 5-y CIR and NRM post TBI were 0.15±0.06 and 0.11±0.05, 0.15±0.06 and 0.11±0.05, and 0.20±0.02 and 0.07±0.02, for the three groups, respectively (p=n.s.). The 3-y GvHD-free/relapse-free survival (GRFS) was similar between HYPO/BCRABL and NEITHER (0.60±0.06 and 0.58±0.03, respectively) and reached a plateau at 5-years (0.60±0.06 and 0.57±0.03, respectively). In subgroup analyses of pts in CR1 and >CR1, comparable outcomes were observed for HYPO, BCRABL, and NEITHER, hence results were not affected by the remission status. BCRABL and NEITHER pts receiving non-TBI conditioning had inferior outcomes (5-y EFS of 0.56±0.10 and 0.47±0.04, respectively) with a high 5-y CIR (0.36±0.09 and 0.43±0.04, respectively) as compared to those who received TBI. Similar 5-y OS and EFS were seen in HYPO pts whether receiving TBI (0.76±0.8 and 0.70±0.08) or chemo conditioning (83±0.09 and 83±0.09). Rates of NRM and acute/chronic graft-versus-host disease were similar in pts receiving TBI/ non-TBI conditioning and in BCRABL/HYPO vs. NEITHER pts. In multivariate analysis adjusted for CR status, conditioning and MRD, EFS did not differ between HYPO/BCRABL and NEITHER pts. Conclusions: Children receiving HSCT for treatment of BCP-ALL with either hypodiploidy or bcr-abl1 fusion showed outcomes comparable to BCP-ALL pts without these genetic lesions. CIR and EFS reached plateaus beyond 3 y post-HSCT, indicating that approximately two-thirds of these pts can be cured by HSCT with low rates of treatment-related mortality. Pts with BCRABL achieved favorable outcomes without the need for long-term or lifelong TKI therapy.

Efficacy and Tolerance of Brexucabtagene Autoleucel in Adults with Relapsed/Refractory B-Cell Precursor Acute Lymphoblastic Leukemia : A Graall Study from the Descar-T Registry

Background : Brexucabtagene autoleucel (brexu-cel) was recently approved for adult patients with relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) based on the results of the ZUMA-3 phase 2 study. After a median follow-up of 39 months, brexu-cel showed a complete remission (CR)/CR with incomplete hematologic recovery (CRi) rate of 71% and a median overall survival of 26 months (Shah B, et al. ASCO 2023). In the present study, we aimed at investigating the efficacy and safety of brexu-cel in a real-life cohort of adult BCP-ALL patients included in the French early access program. Methods : In this Group for Research on Adult Acute Lymphoblastic leukemia (GRAALL) study, we performed an analysis of the DESCAR-T registry which collects real-life data of patients treated with CAR T-cells since July 2018 in France. All patients who underwent a leukapheresis with an intent to manufacture brexu-cel were included (N=80). Efficacy and safety analyses were performed in the 64/66 infused patients with available response assessment after infusion. The data cut-off was June 2023. The present study reports on best overall response, overall survival (OS), relapse-free survival (RFS), as well as immune effector cell-associated neurotoxicity syndrome (ICANS), and cytokine release syndrome (CRS) incidence and grading (according to ASTCT Consensus). Results : Between May 2019 and February 2023, 80 adults with R/R BCP-ALL from 22 French centers had a leukapheresis for brexu-cel. Among them, 14 patients (18%) were not infused because of death (8 pts), progression (1 pt), manufacturing failure (2 pts), or other causes (3 pts). Among the 64 infused patients with available response assessment, median age was 43.5 yrs (range, 22-69). A Philadelphia(Ph)-chromosome was found in 20/64 (31%). Patients had received a median of 3 (range, 1-6) prior lines of treatment including blinatumomab, inotuzumab ozogamicin and allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 67%, 19% and 70% of patients respectively. Twelve patients (19%) had an active CNS disease (CNS2/3) prior to apheresis and 18/40 (45%) had bone marrow blasts ≥ 25% before lymphodepletion (LD). ECOG before infusion was ≥2 in 6/64 (9%) patients. All patients received fludarabine and cyclophosphamide as LD. Median time from apheresis to infusion was 40 days (range, 27-99). CRS and ICANS occurred in 48/64 (77%, grade 3+ in 6%) and 28/64 (45%, grade 3+ in 8%) of patients respectively and were reversible. A CR was achieved in 49/64 patients (77%) of whom 23/25 (92%) were MRD-negative. An older age, a prior allo-HSCT, and the occurrence of CRS were associated with significantly higher CR rates. The median follow-up was 13 months. Three patients were bridged to an allo-HSCT in continuous complete remission (second allo-HSCT for 2 of them). The median RFS and OS were 12.9 months and 15.6 months respectively (Figure). In patients who achieved a CR, median OS was 25.8 months (Figure). Patients with CNS involvement before leukapheresis had a significantly shorter RFS (HR 5.4, 95%CI 1.9-15.8, p=0.002) and OS (HR 3.3, 95%CI 1.4-8.2, p=0.008). Number of prior treatment lines, prior inotuzumab, prior blinatumomab, or pre-LD BM blast% were not associated with outcome. A better OS was observed in patients with prior allo-HSCT (HR .4, 95%CI 0.2-0.9, p=0.04). Conclusion : This multicenter real-life study confirms the efficacy and acceptable safety profile of brexu-cel in adult patients with R/R BCP-ALL. It also suggests that patients with CNS involvement, that were not eligible to ZUMA-3 study, have a limited benefit of brexu-cel when compared to other patients.

Reduced-Dose Chemotherapy Followed By Blinatumomab As Induction Therapy in Treatment of Newly Diagnosed Philadelphia Chromosome-Negative B-Cell Precursor Acute Lymphoblastic Leukemia - Interim Results from a Multicenter, Single-Arm, Phase 2 Study

Introduction: Although intensive pediatric chemotherapeutic regimens have shown good efficacy in the treatment of Philadelphia Chromosome-negative (Ph-negative) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults, the high incidence of chemotherapy-related toxicities limits their use. Blinatumomab, a CD3/CD19 bispecific T-cell antibody, has shown high efficacy in relapsed/refractory or minimal residual disease (MRD) positive BCP-ALL; however, the efficacy of single-agent blinatumomab does not meet the treatment needs of adult patients. Therefore, combining low-intensity chemotherapy with blinatumomab could be a better induction regimen for newly diagnosed adult patients with BCP-ALL. Methods: This multicenter, single-arm, phase 2 trial (NCT05557110) enrolled adult patients (aged 15-59 years) with newly diagnosed Ph-negative BCP-ALL. The induction regimen comprised reduced intensity chemotherapy (idarubicin 8 mg/m 2 on day 1, vindesine 4 mg on day 1, and dexamethasone 9 mg/m 2/day from day 1-7) followed by 2 weeks of blinatumomab (9 ug/day from day 8-14, 28 ug/day from day 15-21). The primary endpoint was to assess the overall response rate (ORR=complete remission [CR] + CR with partial hematologic recovery [CRh] + CR with incomplete hematologic recovery [CRi]) of this induction regimen. Secondary endpoints were MRD negativity (<1x10 -4 by flow cytometry), event-free survival (EFS), overall survival (OS) and safety. Bone marrow evaluation and one intrathecal injection chemotherapy were performed on day 22±2. Patients not achieving ORR continued blinatumomab for another 2 weeks (28 ug/day from day 1-14), followed by bone marrow re-evaluation. Consolidation therapy was given after achieving ORR with recommended multidrug combination chemotherapy (including high-dose methotrexate or cytarabine combined with asparaginase) or alternating with blinatumomab (28 ug/day for 28 days). If allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not performed, consolidation therapy needs at least 4 courses before 2 years of maintenance therapy. Results: Interim results are presented here.By July 6, 2023 (data cut-off), 25 patients were enrolled with a median age of 42 years (range: 15-53); 68% were females. The Eastern Cooperative Oncology Group Performance Status score ranged from 0-1. Eight (32.0%) patients had poor cytogenetic risk factors, including 3 with P2RY8::CRLF2, 2 with hypodiploidy, 2 with KMT2A rearranged and 1 with alterations of IKZF1 (Table 1). The bone marrow evaluation done in 19 patients on day 8 prior to blinatumomab treatment showed that none of the patients had got ORR. All 21 (100%) patients evaluable for the primary endpoint attained CR/Cri - 19 at the end of 2 weeks of blinatumomab and the remaining 2 after another 2 weeks of blinatumomab (Table 2). The MRD negativity rate was 90.5% (19/21) after 2 weeks of blinatumomab which increased to 95.5% (20/21) after another 2 weeks of treatment with blinatumomab. In 21 patients evaluated for safety, blinatumomab-related cytokine release syndrome was reported in 10 (47.6%) patients (grade 1: n=6, grade 2: n=3 and grade 3: n=1). No incidence of neurotoxicity was reported. Febrile neutropenia was reported in 4 (19%) patients (all ≥grade 3). The non-hematological AEs reported were fever in 7 (33.3%) patients (any grade), increased ALT in 10 (47.6%) patients (any grade) and infections in 4 (19%) patients, including pneumonia in 2 and sepsis in 2 (all ≥grade 3). The median duration of neutrophil deficiency (<0.5×10 9/L) and platelet deficiency (<20×10 9/L) during induction therapy was 9 days (range: 0-20) and 1 day (range: 0-18), respectively. The median follow-up time was 4 months and no relapse or leukemia-related deaths were observed. The median EFS and OS were not yet reached. All enrolled patients are alive except one who died of infection after multidrug combination consolidation chemotherapy. The median cycles for consolidation were 2 (range: 1-5) with 3 patients undergoing allo-HSCT. Conclusion: Thesepreliminary results indicate that the reduced intensity chemotherapy followed by blinatumomab is an effective regimen in adults with newly diagnosed Ph-negative BCP-ALL, resulting in favorable response rates, deep remission, and low-grade treatment-related AEs. These promising findings may provide a rationale for integrating blinatumomab into future induction therapy recommendations.

Single-Cell Transcriptomic Profiling of T Cells from Blinatumomab-Treated Patients with B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Reveals Circulating CD8 T Cell Subsets Associated with Treatment Response

Background Blinatumomab (Blina), a bispecific CD3xCD19 T-cell engager (BiTE®), is approved for patients with relapsed/refractory (R/R) and measurable residual disease (MRD) positive B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL). However, a significant percentage of patients do not respond to Blina, and the determinants of resistance are currently unknown. Here, we used Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to identify pre-treatment T cell features associated with response. Methods PBMCs of 13 patients with BCP-ALL who received Blina at the LMU Munich University Hospital from 2016 to 2021 were collected before treatment start. CITE-seq(BD Rhapsody Platform) was performed on a total of 34.907 cells isolated via magnetic-activated cell sorting based on the expression of CD3. An AbSeq panel containing 7 surface markers (CD4, CD8, CCR7, CD45RA, CX3CR1, CD127 and PD1) and a customized T-cell targeted RNA sequencing panel (477 genes) were employed. A Monte-Carlo permutation test was used to compare the proportions of cells in different clusters between Responders (R) and Non-Responders (NR). Significant differences were assumed with a fold change threshold of at least 1.5 (log2FC (LFC) ≥ 0.58) and a false discovery rate (FDR) cutoff of < 0.05. Results We used CITE-seq to characterize T cells in peripheral blood (PB) samples obtained from R/R (n=3) and MRD+ (n=10) BCP-ALL patients. Ten patients (9 MRD+, 1 R/R) responded to the Blina treatment. An integrated strategy based on Reciprocal Principal Component Analysis(RPCA) was implemented and bioinformatic analysis of gene expression mapped in two dimensions via uniform manifold approximation and projection (UMAP) identified 12 CD3 pos distinct clusters. Within CD8 pos T cells of R, we found an increased proportion of CCR7 low Granzyme K(GZMK) high Effector Memory-like cells (GZMK high TEM-like, LFC 0.78, 95CI: 0.66 to 0.92; FDR=0.0012) and a reduced percentage of Naïve cells(CCR7 high LEF1 high CD62L high), when compared to NR (LFC -0.59, 95CI: -0.54 to -0.64; FDR =0.0012, Figure1A). Atrend towards an increased proportion of CCR7 lowCX3CR1 highGZMB high terminal effector-like cells (GZMB high TE-like, LFC 0.57, 95CI: 0.45-0.70; FDR=0.0013) was also noted in CD8 pos T cells of R vs NR. Peripheral CD4 pos T cells of R exhibited a tendency towards an increased proportion of GZMK high cytotoxic T cells (LFC 0.43, 95CI: 0.26 to 0.58; FDR=0.0019) and a reduced percentage of FOXP3 high T regulatory cells (LFC -0.19, 95CI: -0.03 to -0.34; FDR=0.025), compared to NR. Furthermore, we investigated gene expression differences between R and NR cells in each cell cluster. Naïve T cells (CD8 and CD4) of NR showed a significantly increased (LFC>1, FDR<0.05, Figure1B) PRDM1 (Blimp-1) expression, a key promoter of T-cell terminal differentiation that is known to be involved in CD19 CAR-T dysfunction. CD8GZMB high TE-likecells of NR showed upregulated expression of genes involved in chemokine production and cytokine signaling (CCL3, IFNGR1) and downregulated LAIR2, a decoy receptor protecting CD8 T cells from collagen-induced exhaustion. Additionally, TOX, a regulator of T cell exhaustion, was moderately upregulated in CD8GZMB high TE-like cells of NR (LFC 0.41, FDR=0.007). Conclusions Taken together, we were able to identify CD8 T cell subsets that are associated with a response to Blina treatment, as well as gene sets involved in BiTE-mediated T-cell function. Interestingly, the PRDM1 overexpression we found in both CD4 and CD8 naive T cells of Non-Responders has already been linked to T-cell dysfunction in the context of CD19 CAR-T cell therapy. We plan to confirm the results of our analysis in an independent patient cohort. Concomitantly, we will enrich the dataset with results from multi-parameter flow cytometry and in vitro T-cell functional assays conducted using longitudinally collected PB samples from Blina-treated patients. Figure1 A)Log Fold Change(log FC) of the proportion of Responders vs Non Responders CD8 pos cells in each cluster. The dots represent the observed logFC, the horizontal line the bootstrapped 95%Confidence Intervals; PTGDR2= prostaglandin D2 receptor 2; ns= not significant B) Differentially expressed genes in Non Responders vs Responders CD8 pos naïve T cells. FDR=False Discovery Rate; CCL5= Chemokine (C-C motif) ligand 5 ; PRDM1= PR/SET Domain 1;SGK1= serum/glucocorticoid regulated kinase 1.

Update of Olverembatinib (HQP1351) Overcoming Ponatinib and/or Asciminib Resistance in Patients (Pts) with Heavily Pretreated/Refractory Chronic Myeloid Leukemia (CML) and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph + ALL)

Background Olverembatinib, a novel, potent BCR::ABL1 tyrosine kinase inhibitor (TKI), shows strong antitumor activity in pts with CML and Ph + ALL. Here, we report on the safety, efficacy, and pharmacokinetic (PK) profiles of olverembatinib in pts with CML (all phases) and Ph + ALL outside of China, particularly in pts previously treated with ponatinib and/or asciminib. Methods Olverembatinib was administered orally once every other day (QOD) in continuous 28-day cycles. In the monotherapy cohort, pts were enrolled after treatment failure on at least 2 prior TKIs and randomized to olverembatinib QOD 30, 40, or 50 mg. In the combination cohort, pts with Ph + B-cell precursor (BCP) ALL or lymphoid CML-BP (CML-LBP) resistant to at least 1 TKI were enrolled and received olverembatinib (30 or 40 mg) QOD in combination with blinatumomab. Results As of June 30, 2023, 76 pts were enrolled, including 57 with CML-CP and 19 with advanced Ph + leukemia. The median (range) age was 54.5 (21-80) years, and 56.6% of pts were male. In all, 11 (14.5%), 23 (30.3%), and 39 (51.3%) pts had received 2, 3, and ≥ 4 prior TKIs, respectively. A total of 52.6% of pts were previously treated with ponatinib, of whom 67.5% were resistant and 25.0% intolerant to the drug, and 7.5% experienced treatment failure for other reasons. A total of 27.6% of pts were previously treated with asciminib, of whom 71.4% were resistant and 19.1% intolerant to the agent, and 9.5% experienced treatment failure for other reasons. At baseline, 32% of pts had T315I mutations, 38% hypertension, and 17.1% other cardiovascular comorbidities. Median (range) treatment duration was 24.1 (0-134) weeks, and PK profiles were similar to historical PK data on Chinese pts. Twelve pts with CML-CP and 7 with advanced Ph + leukemia discontinued treatment: 4 because of AEs, 7 disease progression, and 8 other reasons. A total of 54 of 65 (83.1%) pts who received ≥ 1 dose of olverembatinib experienced any-grade TRAEs. Grade ≥ 3 AEs occurring in ≥ 3 pts (≥ 4.6% incidence) included thrombocytopenia (17%); neutropenia (13.8%); elevated blood creatine phosphokinase (13.8%); leukopenia (7.7%); and anemia and elevated lipase (4.6% each). Ten (15.4%) pts experienced olverembatinib treatment-related serious AEs, of which each were experienced by 1 (1.5%) pt. Two (3.1%) pts discontinued the study because of TRAEs. No TRAE-associated deaths were reported. Olverembatinib showed sustained antileukemic activity in pts with CML and Ph + ALL (Table 1). Among 50 efficacy-evaluable pts with CML-CP, the rate of complete cytogenetic response (CCyR) was 57% (25/44) and major molecular response (MMR) 43% (21/49). Efficacy improved over time; the MMR rate in pts with CML-CP treated for 6 months was 66% and 88% in pts treated for 12 months. Among pts whose disease failed ≥ 4 prior TKIs, CCyR and MMR rates were 57% (13/23) and 42% (11/26), respectively. In pts with CML-CP harboring the T315I mutation, rates of CCyR and MMR were 60% (9/15) and 44% (7/16), respectively, and 55% (16/29) and 42% (14/33) in pts without the T315I mutation. Among evaluable ponatinib-failed pts, 8/15 (53%) achieved CCyR and 6/16 (38%) MMR. Among pts who failed asciminib therapy, 3/7 (43%) achieved CCyR and 3/8 (38%) MMR. Of 8 pts with CML-CP who had prior exposure to both ponatinib and asciminib, 2 (25%) achieved MMR. At 24 months, PFS was 75% (95% CI, 56.1-86.7) and OS was 97.6% (95% CI, 90.8-99.4). Thirteen pts with advanced Ph + leukemia were efficacy-evaluable, of whom 3 (23%) achieved MMR; only 1 of 3 pts with the T315I mutation achieved MMR; the other 2 were also resistant to ponatinib treatment. The median (95% CI) PFS of efficacy-evaluable pts with advanced leukemia was 12.7 (4-19.5) months. In the combination cohort, 2 pts with Ph + BCP ALL received olverembatinib 30 mg QOD with blinatumomab; both achieved CCyR and 1 achieved a negative MRD status after 1 treatment cycle. Conclusions Olverembatinib alone or combined with blinatumomab was efficacious and well tolerated in pts with heavily pretreated CML or Ph + ALL . Olverembatinib monotherapy was potent in pts who were either resistant or intolerant to ponatinib and/or asciminib, regardless of T315I mutation status. Olverembatinib may provide an effective new treatment option for pts after failure of 2 or more TKIs. Internal study identifier: HQP1351-CU101. Clinicaltrials.gov identifier: NCT04260022.

Role of Microenvironment in Influencing the Activity of CD19 CAR T Cells in B-Cell Precursor Acute Lymphoblastic Leukemia (B-ALL)

Background: As a therapy based on immune cells, CAR T cells treatment elicits an acute response involving both innate and adaptive immunity which is followed by a process of resolution. A major interest is to identify, within the tumor microenvironment (TME), factors that influence the activity and potency of anti-CD19 CAR T cells. The B-ALL TME provides factors that protect leukemia and, conversely, suppress effector T cells. However, studies on the interaction between the bone marrow (BM) TME and CAR T cells are still limited. We hypothesized that the immunological niche of the BM reacts to CAR T-cell-mediated inflammation by modulating the immune response through the activation of inhibitory pathways and molecules. Methods: We conducted an academic phase I/II study (NCT03389035) with donor-derived anti-CD19 CAR T cells generated with Sleeping Beauty transposon (CARCIK-CD19) in B-ALL patients relapsed after alloHSCT. Hematology laboratory data, CAR T,and CD3+ cell kinetics were analyzed in 43 pediatric and adult B-ALL patients who underwent commercial and investigational anti-CD19 CAR T cell treatment. The scRNA-seq libraries of BM samples were generated using Chromium Single Cell 5′ Reagent Kit of the 10x Genomics (v3.1). Validation was performed in 20 pre-/post- CAR-T matched BM samples by a 30-color flow cytometry panel. Results: Following the peak of expansion, CAR T-cell counts decreased in the third and four-week post-infusion. A similar dynamic of expansion and contraction was observed in CD3+ T cells, total white blood cells (WBC), and monocytes, indicating that a widespread resolution of CAR T-driven inflammation occurs during the first month after infusion. Interestingly, the intensity of the response seems to differ depending on the cell product used. To elucidate the mediators involved in the resolution of CAR T-cell-mediated inflammation, the single-cell transcriptome of patients' BM cells at early time points post-CAR T-cell infusion (1-2 months) was compared to pre-treatment samples at the moment of disease relapse and the infusion products. Eighteen sequencing libraries were analyzed, 6 libraries per patient, for a total of 71407 high-quality cells at an average of 65000 reads per cell. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. Notably, extensive integration of the dataset coming from the individual patients was observed.The more representative clusters were classified into infusion product, CD4 and CD8 endogenous population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the composition of BM after CAR T cells infusion compared to pre-treatment samples. Complete disappearance of the B cells associated cluster was observed after treatment. CAR T-cell treatment generated a remodeling of the BM microenvironment, with an increase in the myeloid cells (specifically monocytes), NK, NK-T, and exhausted CD8+ T cell populations. After CAR T-cell infusion, myeloid cells displayed a higher resemblance to myeloid-derived suppressor cells (MDSCs). GeneSet Enrichment Analysis (GSEA) showed significant enrichment in pathways associated with immunosuppression both in the myeloid compartment and in endogenous T cells and in CAR T cells. Of note, the genes involved are also strictly correlated to the generation of terminally exhausted T cells and the emergence of MDSCs. Spectral flow cytometry-based experiments validated our observation of an increase of monocytes, MDSC-like cells, NK, NK-T, and CD8 terminal cells. Modeling intercellular communication using NicheNet suggested the induction of immunosuppressive genes in myeloid cells by both CAR T cells and endogenous T cells. Using the same tool with all niches as the sender and T cells as the receiver, we observed that myeloid cells induced significant signaling in both CAR T cells and endogenous T cells. Conclusions: Through sc-RNAseq and spectral flow cytometry, we have characterized changes in the BM TME after CAR T cell therapy. Specifically, CAR T cells-mediated myeloid activation is associated with pathways of immune dysregulation that may dampen CAR T cell expansion and antagonize the effects of the therapy. Furthermore, these data support the hypothesis that transcriptomic interrogation can infer pathways relevant to the communication between CAR T cells and the TME.