Targeting autophagy overcomes cancer-intrinsic resistance to CAR-T immunotherapy in B-cell malignancies.

BACKGROUND: Chimeric antigen receptor T (CAR-T) therapy has substantially revolutionized the clinical outcomes of patients with hematologic malignancies, but the cancer-intrinsic mechanisms underlying resistance to CAR-T cells remain yet to be fully understood. This study aims to explore the molecular determinants of cancer cell sensitivity to CAR-T cell-mediated killing and to provide a better understanding of the underlying mechanisms and potential modulation to improve clinical efficacy. METHODS: The human whole-genome CRISPR/Cas9-based knockout screening was conducted to identify key genes that enable cancer cells to evade CD19 CAR-T-cell-mediated killing. The in vitro cytotoxicity assays and evaluation of tumor tissue and bone marrow specimens were further conducted to confirm the role of the key genes in cancer cell susceptibility to CAR-T cells. In addition, the specific mechanisms influencing CAR-T cell-mediated cancer clearance were elucidated in mouse and cellular models. RESULTS: The CRISPR/Cas9-based knockout screening showed that the enrichment of autophagy-related genes (ATG3, BECN1, and RB1CC1) provided protection of cancer cells from CD19 CAR-T cell-mediated cytotoxicity. These findings were further validated by in vitro cytotoxicity assays in cells with genetic and pharmacological inhibition of autophagy. Notably, higher expression of the three autophagy-related proteins in tumor samples was correlated with poorer responsiveness and worse survival in patients with relapsed/refractory B-cell lymphoma after CD19 CAR-T therapy. Bulk RNA sequencing analysis of bone marrow samples from B-cell leukemia patients also suggested the clinical relevance of autophagy to the therapeutic response and relapse after CD19 CAR-T cell therapy. Pharmacological inhibition of autophagy and knockout of RB1CC1 could dramatically sensitize tumor cells to CD19 CAR-T cell-mediated killing in mouse models of both B-cell leukemia and lymphoma. Moreover, our study revealed that cancer-intrinsic autophagy mediates evasion of CAR-T cells via the TNF-α-TNFR1 axis-mediated apoptosis and STAT1/IRF1-induced chemokine signaling activation. CONCLUSIONS: These findings confirm that autophagy signaling in B-cell malignancies is essential for the effective cytotoxic function of CAR-T cells and thereby pave the way for the development of autophagy-targeting strategies to improve the clinical efficacy of CAR-T cell immunotherapy.

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression.

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Transcription factor 12-mediated self-feedback regulatory mechanism is required in DUX4 fusion leukaemia.

BACKGROUND: IGH::DUX4 is frequently observed in 4% B-cell acute lymphoblastic leukaemia patients. Regarding the IGH::DUX4-driven transactivation and alternative splicing, which are the main reasons behind this acute leukaemia outbreak, it remains unclear how transcriptional cofactors contribute to this oncogenic process. Further investigation is required to elucidate their specific role in leukaemogenesis. METHODS: In order to investigate the cofactors of IGH::DUX4, integrated mining of Chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing of leukaemia cells and patient samples were conducted. Furthermore, to elucidate the synergistic interaction between transcription factor 12 (TCF12) and IGH::DUX4, knockdown and knockout experiment, mammalian two-hybridisation assay, co-immunoprecipitation and in situ proximity ligation assays were carried out. Additionally, to further investigate the direct interaction between TCF12 and IGH::DUX4, AI-based structural simulations were utilised. Finally, to validate the synergistic role of TCF12 in promoting IGH::DUX4 leukaemia, cell proliferation, apoptosis and drug sensitivity experiments were performed. RESULTS: In this study, we observed that the IGH::DUX4 target gene TCF12 might be an important cofactor/helper for this oncogenic driver. The co-expression of IGH::DUX4 and TCF12 resulted in enhanced DUX4-driven transactivation. Supportively, knockdown and knockout of TCF12 significantly reduced expression of IGH::DUX4-driven target genes in leukaemia REH (a precursor B-cell leukaemia cell line) and NALM-6 cells (a precursor B-cell leukaemia cell line). Consistently, in TCF12 knockout cells, the expression of structure-based TCF12 mutant, but not wild-type TCF12, failed to restore the TCF12-IGH::DUX4 crosstalk and the synergistic transactivation. More importantly, the breakdown in TCF12-IGH::DUX4 cooperation impaired IGH::DUX4-driven leukaemia cell survival, caused sensitivity to the chemotherapy. CONCLUSIONS: Altogether, these results helped to define a previously unrecognised TCF12-mediated positive self-feedback regulatory mechanism in IGH::DUX4 leukaemia, which holds the potential to function as a pivotal drug target for the management of this particular form of leukaemia. HIGHLIGHTS: Transcription factor 12 (TCF12) is a new novel cofactor in IGH::DUX4 transcriptional complexes/machinery. TCF12 mediates a positive self-feedback regulatory mechanism in IGH::DUX4-driven oncogenic transaction. IGH::DUX4-TCF12 structure/cooperation might represent a potent target/direction in future drug design against B-cell acute lymphoblastic leukaemia.

Lessons from mouse models in the impact of risk factors on the genesis of childhood B-cell leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) stands as the primary contributor to childhood cancer-related mortality on a global scale. The development of the most conventional forms of this disease has been proposed to be conducted by two different steps influenced by different types of risk factors. The first step is led by a genetic insult that is presumably acquired before birth that transforms a healthy cell into a preleukemic one, which is maintained untransformed until the second step takes place. This necessary next step to leukemia development will be triggered by different risk factors to which children are exposed after birth. Murine models that recap the stepwise progression of B-ALL have been instrumental in identifying environmental and genetic factors that contribute to disease risk. Recent evidence from these models has demonstrated that specific environmental risk factors, such as common infections or gut microbiome dysbiosis, induce immune stress, driving the transformation of preleukemic cells, and harboring genetic alterations, into fully transformed leukemic cells. Such models serve as valuable tools for investigating the mechanisms underlying preleukemic events and can aid in the development of preventive approaches for leukemia in child. Here, we discuss the existing knowledge, learned from mouse models, of the impact of genetic and environmental risk factors on childhood B-ALL evolution and how B-ALL prevention could be reached by interfering with preleukemic cells.

Genomic, transcriptomic and epigenomic sequencing data of the B-cell leukemia cell line REH.

OBJECTIVES: The aim of this data paper is to describe a collection of 33 genomic, transcriptomic and epigenomic sequencing datasets of the B-cell acute lymphoblastic leukemia (ALL) cell line REH. REH is one of the most frequently used cell lines for functional studies of pediatric ALL, and these data provide a multi-faceted characterization of its molecular features. The datasets described herein, generated with short- and long-read sequencing technologies, can both provide insights into the complex aberrant karyotype of REH, and be used as reference datasets for sequencing data quality assessment or for methods development. DATA DESCRIPTION: This paper describes 33 datasets corresponding to 867 gigabases of raw sequencing data generated from the REH cell line. These datasets include five different approaches for whole genome sequencing (WGS) on four sequencing platforms, two RNA sequencing (RNA-seq) techniques on two different sequencing platforms, DNA methylation sequencing, and single-cell ATAC-sequencing.

Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL.

Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.

Co-opting signalling molecules enables logic-gated control of CAR T cells.

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

ETS Fight Club on Microsatellite Enhancers.

In this issue of Blood Cancer Discovery, Kodgule, Goldman, Monovichet al. cleverly analyzed the transcription regulatory elements to investigate why the second copy of ETV6 is often lost in ETV6::RUNX1-translocated in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). It turns out that ETV6 suppresses the enhancer activity of GGAA microsatellite repeats, preventing ERG from subverting them to activate aberrant oncogene transcription. See related article by Kodgule, Goldman, Monovich et al., p. 34 (5).

Circulating CD22+/CD19-/CD24- progenitors and CD22+/CD19+/CD24- mature B cells: Diagnostic pitfalls for minimal residual disease detection in B-lymphoblastic leukemia.

BACKGROUND: Multiparametric flow cytometry (MFC) has become a powerful tool in minimal residual disease (MRD) detection in B-lymphoblastic leukemia/lymphoma (B-ALL). In the setting of targeted immunotherapy, B-ALL MRD detection often relies on alterative gating strategies, such as the utilization of CD22 and CD24. It is important to depict the full diversity of normal cell populations included in the alternative B-cell gating methods to avoid false-positive results. We describe two CD22-positive non-neoplastic cell populations in the peripheral blood (PB), including one progenitor population of uncertain lineage and one mature B-cell population, which are immunophenotypic mimics of B-ALL. METHODS: Using MFC, we investigated the prevalence and phenotypic profiles of both CD22-positive populations in 278 blood samples from 52 patients with B-ALL; these were obtained pre- and post-treatment with CD19 and/or CD22 CAR-T therapies. We further assessed whether these two populations in the blood were exclusively associated with B-ALL or recent anticancer therapies, by performing the same analysis on patients diagnosed with other hematological malignancies but in long-term MRD remission. RESULTS: The progenitor population and mature B-cell population were detected at low levels in PB of 61.5% and 44.2% of B-ALL patients, respectively. Both cell types showed distinctive and highly consistent antigen expression patterns that are reliably distinguishable from B-ALL. Furthermore, their presence is not restricted solely to B-ALL or recent therapy. CONCLUSIONS: Our findings aid in building a complete immunophenotypic profile of normal cell populations in PB, thereby preventing misdiagnosis of B-ALL MRD and inappropriate management.

B-lymphoblastic leukemia with transient spontaneous remission in the setting of severe group A streptococcus infection.

Spontaneous remission of B-lymphoblastic leukemia (B-ALL) in the setting of viral and bacterial infections has been reported. Here, we present a case of B-ALL that showed a complete remission in the setting of group A streptococcal bacteremia. The patient was an 11-year-old boy who presented with a sore throat, right ear pain, and rhinorrhea. Prior to the diagnosis of B-ALL, he was diagnosed with streptococcal pharyngitis and received a single dose of dexamethasone and azithromycin. One day later, he was found to be pancytopenic and an immunophenotypically abnormal B-lymphoblastic population was detected comprising 0.6% and 16.8% of the peripheral blood and bone marrow cells, respectively. Though a diagnosis of B-ALL was highly suspected, blast percentage was <20% and the bone marrow showed relatively unremarkable trilineage hematopoiesis. On close monitoring, the suspected neoplastic population became undetectable by day 17 and the patient's complete blood count (CBC) completely normalized by day 46. On day 82, a peripheral blood smear demonstrated circulating blasts. Flow cytometry of a bone marrow aspirate revealed B-lymphoblastic leukemia accounting for 94% nucleated cells, consistent with the diagnosis of B-lymphoblastic leukemia. This case is of interest as less than 20 examples of spontaneous remission of B-ALL have been reported in the literature. As the case reported here relapsed and previously reported spontaneously remitting cases have uniformly relapsed, cases of B-ALL with spontaneous remission should be followed very closely for recurrence.

Knockdown of Bcl-3 alleviates psoriasis and dyslipidemia comorbidity by regulating Akt pathway.

BACKGROUND: Psoriasis is considered as an inflammatory skin disease accompanied by dyslipidemia comorbidity. B-cell leukemia-3 (Bcl-3) belongs to IκB (inhibitor of nuclear factor kappa B [NF-κB]) family, and regulates inflammatory response through associating with NF-κB. The role of Bcl-3 in psoriasis was investigated in this study. METHODS: Apolipoprotein E (ApoE)-deficient mice were treated with imiquimod to induce psoriasis and dyslipidemia. Mice were injected intradermally in the back with lentiviral particles encoding Bcl-3 small hairpin RNA (shRNA). Hematoxylin and eosin were used to detect pathological characteristics. The blood lipid levels were determined by automatic biochemical analyzer, and inflammation was assessed by enzyme-linked-immunosorbent serologic assay and real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Bcl-3 was elevated in imiquimod-induced ApoE-deficient mice. Injection with lentiviral particles encoding Bcl-3 shRNA reduced Psoriasis area and severity index (PASI) score in ApoE-deficient psoriatic mice. Knockdown of Bcl-3 also ameliorated imiquimod-induced psoriasiform skin lesions in ApoE-deficient mice. Moreover, loss of Bcl-3 enhanced expression of loricrin, an epidermal barrier protein, reduced expression of proliferating cell nuclear antigen (PCNA) and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) in imiquimod-induced ApoE-deficient mice. The enhanced levels of blood lipid in ApoE-deficient mice were attenuated by silencing of Bcl-3 with increase of high-density lipoprotein, and reduction of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Knockdown of Bcl-3 attenuated imiquimod-induced decrease of transforming growth factor beta (TGF-ß), and increase of Interleukin (IL)-17A, IL-23, IL-6, and tumor necrosis factor-α (TNF-α) in ApoE-deficient mice. Protein expression of phospho-Akt (p-Akt) and p-GSK3ß in ApoE-deficient psoriatic mice was decreased by silencing of Bcl-3. CONCLUSION: Loss of Bcl-3 exerted anti-inflammatory effect on psoriasis and dyslipidemia comorbidity through inactivation of Akt/GSK3ß pathway.

Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia.

Genome editing of allogeneic T cells can provide "off-the-shelf" alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator-like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3' long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 106 to 2.0 × 106 CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.

Clinicopathologic and genetic evaluation of B-lymphoblastic leukemia with intrachromosomal amplification of chromosome 21 (iAMP21) in adult patients.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a rare provisional entity of B-cell acute lymphoblastic leukemia (B-ALL) in the current WHO classification and has been described as specific for pediatric patients with a median age at diagnosis of 9-10 years. We report two adult cases of B-ALL with iAMP21, one 31-year-old woman and one 40-year-old man, identified by karyotyping and next generation sequencing (NGS), with fluorescence in situ hybridization (FISH) pattern meeting diagnostic criteria for iAMP21. Both patients were treated on high-risk chemotherapeutic regimen followed by stem cell transplant. In contrast to reported high relapse rate within the first three years in pediatric population, our adult patients are alive in remission, with the interval from diagnosis to last follow up of 2.95 and 3.96 years. Our cases illustrate the importance of screening for iAMP21 in adult population when ETV6-RUNX1 FISH testing is not routinely performed for adult patients.

Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

CAR-T Cells Shoot for New Targets: Novel Approaches to Boost Adoptive Cell Therapy for B Cell-Derived Malignancies.

Chimeric antigen receptor (CAR)-T cell therapy is undeniably a promising tool in combating various types of hematological malignancies. However, it is not yet optimal and a significant number of patients experience a lack of response or relapse after the treatment. Therapy improvement requires careful analysis of the occurring problems and a deeper understanding of the reasons that stand behind them. In this review, we summarize the recent knowledge about CAR-T products' clinical performance and discuss diversified approaches taken to improve the major shortcomings of this therapy. Especially, we prioritize the challenges faced by CD19 CAR-T cell-based treatment of B cell-derived malignancies and revise the latest insights about mechanisms mediating therapy resistance. Since the loss of CD19 is one of the major obstacles to the success of CAR-T cell therapy, we present antigens that could be alternatively used for the treatment of various types of B cell-derived cancers.

Targeting CD10 on B-Cell Leukemia Using the Universal CAR T-Cell Platform (UniCAR).

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

[A retrospective comparative study of haplotype hematopoietic stem cell transplantation and human leukocyte antigen-matched sibling donor hematopoietic stem cell transplantation in the treatment of acute B-lymphocyte leukemia].

Objective: To investigate whether haplotype hematopoietic stem cell transplantation (haplo-HSCT) is effective in the treatment of pre transplant minimal residual disease (Pre-MRD) positive acute B lymphoblastic leukemia (B-ALL) compared with HLA- matched sibling donor transplantation (MSDT) . Methods: A total of 998 patients with B-ALL in complete remission pre-HSCT who either received haplo-HSCT (n=788) or underwent MSDT (n=210) were retrospectively analyzed. The pre-transplantation leukemia burden was evaluated according to Pre-MRD determinedusing multiparameter flow cytometry (MFC) . Results: Of these patients, 997 (99.9% ) achieved sustained, full donor chimerism. The 100-day cumulative incidences of neutrophil engraftment, platelet engraftment, and grades Ⅱ-Ⅳ acute graft-versus-host disease (GVHD) were 99.9% (997/998) , 95.3% (951/998) , and 26.6% (95% CI 23.8% -29.4% ) , respectively. The 3-year cumulative incidence of total chronic GVHD was 49.1% (95% CI 45.7% -52.4% ) . The 3-year cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) of the 998 cases were 17.3% (95% CI 15.0% -19.7% ) and 13.8% (95% CI 11.6% -16.0% ) , respectively. The 3-year probabilities of leukemia-free survival (LFS) and overall survival (OS) were 69.1% (95% CI 66.1% -72.1% ) and 73.0% (95% CI 70.2% -75.8% ) , respectively. In the total patient group, cases with positive Pre-MRD (n=282) experienced significantly higher CIR than that of subjects with negative Pre-MRD [n=716, 31.6% (95% CI 25.8% -37.5% ) vs 14.3% (95% CI 11.4% -17.2% ) , P<0.001]. For patients in the positive Pre-MRD subgroup, cases treated with haplo-HSCT (n=219) had a lower 3-year CIR than that of cases who underwent MSDT [n=63, 27.2% (95% CI 21.0% -33.4% ) vs 47.0% (95% CI 33.8% -60.2% ) , P=0.002]. The total 998 cases were classified as five subgroups, including cases with negative Pre-MRD group (n=716) , cases with Pre-MRD<0.01% group (n=46) , cases with Pre-MRD 0.01% -<0.1% group (n=117) , cases with Pre-MRD 0.1% -<1% group (n=87) , and cases with Pre-MRD≥1% group (n=32) . For subjects in the Pre-MRD<0.01% group, haplo-HSCT (n=40) had a lower CIR than that of MSDT [n=6, 10.0% (95% CI 0.4% -19.6% ) vs 32.3% (95% CI 0% -69.9% ) , P=0.017]. For patients in the Pre-MRD 0.01% -<0.1% group, haplo-HSCT (n=81) also had a lower 3-year CIR than that of MSDT [n=36, 20.4% (95% CI 10.4% -30.4% ) vs 47.0% (95% CI 29.2% -64.8% ) , P=0.004]. In the other three subgroups, the 3-year CIR was comparable between patients who underwent haplo-HSCT and those received MSDT. A subgroup analysis of patients with Pre-MRD<0.1% (n=163) was performed, the results showed that cases received haplo-HSCT (n=121) experienced lower 3-year CIR [16.0% (95% CI 9.4% -22.7% ) vs 40.5% (95% CI 25.2% -55.8% ) , P<0.001], better 3-year LFS [78.2% (95% CI 70.6% -85.8% ) vs 47.6% (95% CI 32.2% -63.0% ) , P<0.001] and OS [80.5% (95% CI 73.1% -87.9% ) vs 54.6% (95% CI 39.2% -70.0% ) , P<0.001] than those of MSDT (n=42) , but comparable in 3-year NRM [5.8% (95% CI 1.6% -10.0% ) vs 11.9% (95% CI 2.0% -21.8% ) , P=0.188]. Multivariate analysis showed that haplo-HSCT was associated with lower CIR (HR=0.248, 95% CI 0.131-0.472, P<0.001) , and superior LFS (HR=0.275, 95% CI 0.157-0.483, P<0.001) and OS (HR=0.286, 95% CI 0.159-0.513, P<0.001) . Conclusion: Haplo HSCT has a survival advantage over MSDT in the treatment of B-ALL patients with pre MRD<0.1% .