Safety and efficacy of co-administration of CD19 and CD22 CAR-T cells in children with B-ALL relapse after CD19 CAR-T therapy.

BACKGROUND: CD19-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown remarkable efficacy in treating relapsed or refractory pediatric B-lineage acute lymphoblastic leukemia (B-ALL). However, poor results are obtained when the same product is reused in patients who relapse after CAR-T. Therefore, there is a need to explore the safety and efficacy of co-administration of CD19- and CD22-targeted CAR-T as a salvage second CAR-T therapy (CART2) in B-ALL patients who relapse after their first CD19 CAR-T treatment (CART1). METHODS: In this study, we recruited five patients who relapsed after CD19-targeted CAR-T. CD19- and CD22-CAR lentivirus-transfected T cells were cultured separately and mixed before infusion in an approximate ratio of 1:1. The total dose range of CD19 and CD22 CAR-T was 4.3 × 106-1.5 × 107/kg. Throughout the trial, we evaluated the patients' clinical responses, side effects, and the expansion and persistence of CAR-T cells. RESULTS: After CART2, all five patients had minimal residual disease (MRD)-negative complete remission (CR). The 6- and 12-month overall survival (OS) rates were 100%. The median follow-up time was 26.3 months. Three of the five patients bridged to consolidated allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CART2 and remained in MRD-negative CR at the cut-off time. In patient No. 3 (pt03), CAR-T cells were still detected in the peripheral blood (PB) at 347 days post-CART2. Cytokine release syndrome (CRS) only occurred with a grade of ≤ 2, and no patients experienced symptoms of neurologic toxicity during CART2. CONCLUSIONS: Mixed infusion of CD19- and CD22-targeted CAR-T cells is a safe and effective regimen for children with B-ALL who relapse after prior CD19-targeted CAR-T therapy. Salvage CART2 provides an opportunity for bridging to transplantation and long-term survival. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000032211. Retrospectively registered: April 23, 2020.

[Clinical effect of the SCMC APL-2010 regimen in treatment of acute promyelocytic leukemia in children: an analysis of 44 cases].

OBJECTIVE: To study the clinical effect of the SCMC APL-2010 regimen in the treatment of acute promyelocytic leukemia (APL) in children. METHODS: A retrospective analysis was performed for the clinical data of 44 children with APL who received treatment with the SCMC APL-2010 regimen between April 2010 and July 2016. The Kaplan-Meier survival analysis was used to evaluate event-free survival (EFS) rate and overall survival (OS) rate. RESULTS: Of the 44 children with APL, 42 (95%) achieved a complete remission (CR) after one course of treatment and 1 achieved CR after two courses of treatment, with an overall CR rate of 98%. The 9-year EFS and OS rates were 96%±3% and 97.7%±2.2% respectively. As for adverse events, 41 (93%) had infection, 29 (66%) had granulocyte reduction, 12 (27%, 1 died) had differentiation syndrome, 16 (36%) had liver dysfunction, 12 (27%) had adverse gastrointestinal reactions, and 7 (16%) had QT prolongation, 1 (2%) had orchitis, and no secondary neoplasm was observed. CONCLUSIONS: Children with APL receiving the SCMC APL-2010 regimen have a good prognosis and can achieve a long-term survival, while treatment-related infection is commonly seen.

Long-term results of the risk-stratified treatment of childhood acute lymphoblastic leukemia in China.

Long-term follow-up data for childhood acute lymphoblastic leukemia (ALL) are scarce in China because of lacking population-based and hospitalized registry system. This retrospective study, conducted at Shanghai's Children's Medical Center in China (SCMC), aimed to investigate the long-term results of childhood ALL and to identify prognostic factors. The Pediatric Oncology Network Database, designed by St. Jude Children's Research Hospital, USA, were used to collect data for the enrolled patients starting in 2005. From 2005 to 2014, 1085 evaluable patients with ALL aged 1 to 18 years old were enrolled and treated using SCMC-ALL-2005 risk-stratified protocol. Complete remission was achieved in 95.6% of patients. At 5 and 10 years, the event-free survival rate was 68.3 ± 1.4% and 64.6 ± 1.6%, and the overall survival rate was 80.0 ± 1.2% and 76.3 ± 1.6%, respectively. The 5-year event-free survival rates were 81.8 ± 2.0%, 67.0 ± 1.9%, and 14.3 ± 4.0% for patients in low-risk, intermediate-risk, and high-risk groups, respectively. The cumulative risk of relapse was 24.5% at 10 years. Induction failure conferred worse prognosis. Patients younger than 1 year of age at diagnosis, intermediate-risk/high-risk group, male gender, and positive minimal residual disease (MRD) results at day 55, both in the univariate and multivariate analysis, were associated with significantly worse prognosis (P < .05). Patients with positive MRD at both day 35 and day 55 were related to a significantly poor outcome (P < .0001), but not for patients with negitive MRD at day 35. The overall outcomes for ALL patients treated with protocol SCMC-ALL-2005 in SCMC are lower than in developed countries. Factors including age, gender, risk group and MRD results at day 55 were associated with treatment outcomes in childhood ALL.

Relatively favorable prognosis for MLL-rearranged childhood acute leukemia with reciprocal translocations.

BACKGROUND: Mixed-lineage leukemia (MLL) with multifarious partner genes leads to aggressive leukemia with dismal outcomes. METHODS: Using panel-based targeted sequencing, we examined 90 cases with MLL-rearranged (MLL-r) childhood acute leukemia, including 55 with acute lymphoblastic leukemia (ALL) and 35 with acute myeloid leukemia (AML). RESULTS: MLL breakpoints and complete rearrangements were identified. A total of 37.8% (34/90) of patients displayed a single direct MLL fusion gene, 15.6% (14/90) carried a single reciprocal fusion, and 27.8% (25/90) had both reciprocal MLL fusion alleles. The remaining 17 MLL-r cases exhibited complex translocations with homozygous disruptions on chromosome 11 or two breakpoints on the same MLL allele with a deletion of functional regions. A total of 77 patients (45 ALL and 32 AML) received chemotherapy with a median follow-up of 2.5 years. Unexpectedly, we identified children with reciprocal MLL fusions who exhibited relatively favorable outcomes compared with those in children with complex translocations or a single direct MLL fusion allele (66.1% vs. 24.6% and 27.6%, P = 0.001). Reciprocal MLL fusion may be functionally rescued by a partially truncated MLL protein. CONCLUSION: Comprehensive MLL-r analysis by targeted next-generation sequencing can provide detailed molecular information and is helpful for precise stratified treatment and clinical prognosis determination.

[Analysis of clinical manifestation and genetic mutation in a child with X-linked chondrodysplasia punctata 2].

OBJECTIVE: To analyze clinical manifestations and genetic mutation in a child with severe short stature and other malformations. METHODS: The child has undergone history taking and physical examination. Genome DNA was extracted from peripheral blood samples of the proband and her family members. Candidate genes were captured with Agilent SureSelect and sequenced on an Illumina platform. Suspected mutation was verified by Sanger sequencing. RESULTS: The patient, a six-year-and-10-month old girl, presented with non-symmetrical short stature, dysmorphism, abnormalities of limbs and spine, amblyopia of left eye, and cataract of right eye, in addition with frequent respiratory infection and micturition. Laboratory testing suggested 25-hydroxy vitamin D deficiency (18.9 ng/mL). Spine X-ray showed multiple malformations with centrums. Her mother also featured short stature (138 cm). Her aunt had short stature (130 cm) and limb-length discrepancy. Her little brother was 2.5 years old, and his height was 81 cm (-3.4 SD). Exome sequencing revealed a heterozygous mutation c.184C to T (p.Arg62Trp) in the proband and her mother. The same mutation was not found in her father and brother. CONCLUSION: The patient was diagnosed with X-linked chondrodysplasia punctata 2. Mutation of the EBP gene probably underlied the disease in this family.

Functional Neural Networks in Human Brain Organoids.

Human brain organoids are 3-dimensional brain-like tissues derived from human pluripotent stem cells and hold promising potential for modeling neurological, psychiatric, and developmental disorders. While the molecular and cellular aspects of human brain organoids have been intensively studied, their functional properties such as organoid neural networks (ONNs) are largely understudied. Here, we summarize recent research advances in understanding, characterization, and application of functional ONNs in human brain organoids. We first discuss the formation of ONNs and follow up with characterization strategies including microelectrode array (MEA) technology and calcium imaging. Moreover, we highlight recent studies utilizing ONNs to investigate neurological diseases such as Rett syndrome and Alzheimer's disease. Finally, we provide our perspectives on the future challenges and opportunities for using ONNs in basic research and translational applications.

Human brain organoids for understanding substance use disorders.

Substance use disorders (SUDs) are complex mental health conditions involving a problematic pattern of substance use. Challenges remain in understanding their neural mechanisms, which are likely to lead to improved SUD treatments. Human brain organoids, brain-like 3D in vitro cultures derived from human stem cells, show unique potential in recapitulating the response of a developing human brain to substances. Here, we review the recent progress in understanding SUDs using human brain organoid models focusing on neurodevelopmental perspectives. We first summarize the background of SUDs in humans. Moreover, we introduce the development of various human brain organoid models and then discuss current progress and findings underlying the abuse of substances like nicotine, alcohol, and other addictive drugs using organoid models. Furthermore, we review efforts to develop organ chips and microphysiological systems to engineer better human brain organoids for advancing SUD studies. Lastly, we conclude by elaborating on the current challenges and future directions of SUD studies using human brain organoids.

Engineering human midbrain organoid microphysiological systems to model prenatal PFOS exposure.

Perfluorooctane sulfonate (PFOS), a class of synthetic chemicals detected in various environmental compartments, has been associated with dysfunctions of the human central nervous system (CNS). However, the underlying neurotoxicology of PFOS exposure is largely understudied due to the lack of relevant human models. Here, we report bioengineered human midbrain organoid microphysiological systems (hMO-MPSs) to recapitulate the response of a fetal human brain to multiple concurrent PFOS exposure conditions. Each hMO-MPS consists of an hMO on a fully 3D printed holder device with a perfusable organoid adhesion layer for enhancing air-liquid interface culturing. Leveraging the unique, simply-fabricated holder devices, hMO-MPSs are scalable, easy to use, and compatible with conventional well-plates, and allow easy transfer onto a multiple-electrode array (MEA) system for plug-and-play measurement of neural activity. Interestingly, the neural activity of hMO-MPSs initially increased and subsequently decreased by exposure to a concentration range of 0, 30, 100, to 300 µM of PFOS. Furthermore, PFOS exposure impaired neural development and promoted neuroinflammation in the engineered hMO-MPSs. Along with PFOS, our platform is broadly applicable for studies toxicology of various other environmental pollutants.

Bioengineering of a human physiologically relevant microfluidic blood-cerebrospinal fluid barrier model.

The human blood-cerebrospinal fluid barrier (hBCSFB) plays a crucial role in regulating brain interstitial fluid homeostasis, and disruption of the hBCSFB is associated with various neurological diseases. Generation of a BCSFB model with human physiologically relevant structural and functional features is crucial to reveal the cellular and molecular basis of these diseases and discover novel neurologic therapeutic agents. Unfortunately, thus far, few humanized BCSFB models are available for basic and preclinical research. Here, we demonstrate a bioengineered hBCSFB model on a microfluidic device constructed by co-culturing primary human choroid plexus epithelial cells (hCPECs) and human brain microvascular endothelial cells (hBMECs) on the two sides of a porous membrane. The model reconstitutes tight junctions of the hBCSFB and displays a physiologically relevant molecular permeability. Using this model, we further generate a neuropathological model of the hBCSFB under neuroinflammation. Overall, we expect that this work will offer a high-fidelity hBCSFB model for studying neuroinflammation-related diseases.

Rational Design and Acoustic Assembly of Human Cerebral Cortex-Like Microtissues from hiPSC-Derived Neural Progenitors and Neurons.

Development of biologically relevant and clinically relevant human cerebral cortex models is demanded by mechanistic studies of human cerebral cortex-associated neurological diseases and discovery of preclinical neurological drug candidates. Here, rational design of human-sourced brain-like cortical tissue models is demonstrated by reverse engineering and bionic design. To implement this design, the acoustic assembly technique is employed to assemble hiPSC-derived neural progenitors and neurons separately in a label-free and contact-free manner followed by subsequent neural differentiation and culture. The generated microtissues encapsulate the neuronal microanatomy of human cerebral-cortex tissue that contains six-layered neuronal architecture, a 400-µm interlayer distance, synaptic connections between interlayers, and neuroelectrophysiological transmission. Furthermore, these microtissues are infected with herpes simplex virus type I (HSV-1) virus, and the HSV-induced pathogenesis associated with Alzheimer's disease is determined, including neuron loss and the expression of Aß. Overall, a high-fidelity human-relevant in vitro histotypic model is provided for the cerebral cortex, which will facilitate wide applications in probing the mechanisms of neurodegenerative diseases and screening the candidates for neuroprotective agents.

Association between body mass index at diagnosis and outcomes in Chinese children with newly diagnosed acute lymphoblastic leukemia.

PURPOSE: Studies of the association between body mass index (BMI) at diagnosis and treatment outcome in children with acute lymphoblastic leukemia (ALL) have yielded inconsistent results. Hence, we conducted a retrospective study in a large cohort of Chinese children with ALL treated with contemporary protocols. PATIENTS AND METHODS: A total of 1437 children (62.1% male; median age at diagnosis 5.7 years, range: 2.3-16.3 years) were enrolled in two consecutive clinical trials at the Shanghai Children's Medical Center. The rates of overall survival, event-free survival, relapse, treatment-related mortality, and adverse events were compared among patients who were underweight (BMI < 5th percentile), at a healthy weight (5th to 85th percentile), overweight (>85th to <95th percentile), and obese (≥95th percentile). RESULTS: At diagnosis, 91 (6.3%) patients were underweight, 1070 (74.5%) were at a healthy weight, 91 (6.3%) were overweight, and 185 (12.9%) were obese. No significant association was found between weight status and 5-year overall survival, event-free survival, or relapse in the overall cohort. When analyzed as a continuous variable, a higher BMI Z-score was associated with treatment-related mortality (hazard ratio 1.33 (95% confidence interval [CI], 1.05-1.68%), p = 0.02). The treatment-related mortality rate was higher in the overweight (5.5%, 95% CI 0.8-10.2%) and obese (3.2%, 95% CI 0.6-5.8%) groups compared with the underweight (0.0%) and healthy-weight groups (1.9%, 95% CI 1.1-2.7%; p = 0.04). Multivariable analysis showed that children who were overweight had a higher risk of treatment-related mortality (hazard ratio 3.8, 95% CI 1.3-11.4). CONCLUSION: While body weight status was not associated with event-free survival or overall survival, overweight patients were at higher risk of treatment-related mortality.

Coadministration of CD19- and CD22-Directed Chimeric Antigen Receptor T-Cell Therapy in Childhood B-Cell Acute Lymphoblastic Leukemia: A Single-Arm, Multicenter, Phase II Trial.

PURPOSE: We determined the safety and efficacy of coadministration of CD19- and CD22-chimeric antigen receptor (CAR) T cells in patients with refractory disease or high-risk hematologic or isolated extramedullary relapse of B-acute lymphoblastic leukemia. PATIENTS AND METHODS: This phase II trial enrolled 225 evaluable patients age ≤ 20 years between September 17, 2019, and December 31, 2021. We first conducted a safety run-in stage to determine the recommended dose. After interim analysis of the first 30 patients treated (27 at the recommended dose) showing that the treatment was safe and effective, the study enrolled additional patients according to the study design. RESULTS: Complete remission was achieved in 99.0% of the 194 patients with refractory leukemia or hematologic relapse, all negative for minimal residual disease. Their overall 12-month event-free survival (EFS) was 73.5% (95% CI, 67.3 to 80.3). Relapse occurred in 43 patients (24 with CD19+/CD22+ relapse, 16 CD19-/CD22+, one CD19-/CD22-, and two unknown). Consolidative transplantation and persistent B-cell aplasia at 6 months were associated with favorable outcomes. The 12-month EFS was 85.0% (95% CI, 77.2 to 93.6) for the 78 patients treated with transplantation and 69.2% (95% CI, 60.8 to 78.8) for the 116 nontransplanted patients (P = .03, time-dependent covariate Cox model). All 25 patients with persistent B-cell aplasia at 6 months remained in remission at 12 months. The 12-month EFS for the 20 patients with isolated testicular relapse was 95.0% (95% CI, 85.9 to 100), and for the 10 patients with isolated CNS relapse, it was 68.6% (95% CI, 44.5 to 100). Cytokine release syndrome developed in 198 (88.0%) patients, and CAR T-cell neurotoxicity in 47 (20.9%), resulting in three deaths. CONCLUSION: CD19-/CD22-CAR T-cell therapy achieved relatively durable remission in children with relapsed or refractory B-acute lymphoblastic leukemia, including those with isolated or combined extramedullary relapse.[Media: see text].

[Evaluation on protocol SCMS-ALL-2005 for childhood B lineage acute lymphoblastic leukemia].

OBJECTIVE: To reduce the risk of therapy related complication during the treatment and keeps the long term event free survival, and to evaluate the results and risk factors of SCMC-lymphoblastic leukemia (ALL)-2005 protocol. METHODS: Designed the new protocol SCMC-ALL-2005 based on the previous protocol XH-99 for ALL. Divided the patients into low, median and high risk groups depends on risk factors including day 33 and 55 minimal residual disease (MRD) level. The higher risk group, the more intensive therapy was given. All the cases were registed on pediatric oncology network database (POND). All the abandonment patients were counted as event. From May 1(st) 2005 to April 30(th) 2009, 351 children who were newly diagnosed as B lineage ALL were enrolled in this study. The prognoses relating to risk grouping, age, mutation gene and MRD level were analyzed. RESULTS: Up to June 30, 2011, 273 patients were followed up with median time 49 months (range 26 to 74 months). Three hundred and forty-five patients (98.29%) achieved complete remission on day 35 induction. 12 cases were younger than 1 year old (3.42%), 285 cases between 1 and 9 years old (81.20%), 54 cases 10 to 18 years old (15.38%). Five year event-free survival (EFS) was 34%, 72% and 63%, respectively. One hundred and fifty-six cases belonged to lowered risk (44.44%), 177 to middle risk (50.43%) and 18 to higher risk (5.13%). Five year EFS was 78%, 64% and 30%, respectively. In this study, 18 patients were detected positive for BCR/ABL, 3 for MLL/AF4, 16 for PBX/E2A, and 36 for TEL/AML. The 5 year EFS were 11%, 66%, 75% and 74%, respectively. A total of 300 cases were tested for MRD levels on day 35. Of them, 241 cases were with MRD ≤ 0.01% (negative), and 59 cases > 0.01% (positive). The 5 year relapse free survival (RFS) was 79% and 58%, respectively. Total 6 patients died of complication (1.71%). 18 patients were abundant treatment with no disease progress. 70 patients relapsed (19.94%), including 52 bone marrow, 8 central nerve system (CNS), 1 both in bone marrow and CNS, 1 second cancer (M(4)) and 8 testis. Five year overall survival (OS) and EFS are 84% and 69%. CONCLUSIONS: The risk of therapy related death is low with the protocol SCMC-ALL-2005. MRD affects the prognosis. The long term prognosis is poor for high risk group, with BCR/ABL and positive MRD.

High-throughput bioengineering of homogenous and functional human-induced pluripotent stem cells-derived liver organoids via micropatterning technique.

Human pluripotent stem cell-derived liver organoids are emerging as more human-relevant in vitro models for studying liver diseases and hepatotoxicity than traditional hepatocyte cultures and animal models. The generation of liver organoids is based on the Matrigel dome method. However, the organoids constructed by this method display significant heterogeneity in their morphology, size, and maturity. Additionally, the formed organoid is randomly encapsulated in the Matrigel dome, which is not convenient for in situ staining and imaging. Here, we demonstrate an approach to generate a novel type of liver organoids via micropatterning technique. This approach enables the reproducible and high-throughput formation of bioengineered fetal liver organoids with uniform morphology and deterministic size and location in a multiwell plate. The liver organoids constructed by this technique closely recapitulate some critical features of human liver development at the fetal stage, including fetal liver-specific gene and protein expression, glycogen storage, lipid accumulation, and protein secretion. Additionally, the organoids allow whole-mount in-situ staining and imaging. Overall, this new type of liver organoids is compatible with the pharmaceutical industry's widely-used preclinical drug discovery tools and will facilitate liver drug screening and hepatotoxic assessment.

Size- and density-dependent acoustic differential bioassembly of spatially-defined heterocellular architecture.

Emerging acoustic bioassembly represents an attractive strategy to build cellular closely-packed organotypic constructs in a tunable manner for biofabrication. However, simultaneously assemble heterogeneous cell types into heterocellular functional units with spatially-defined cell arrangements, such as complementary and sandwich cytoarchitectures, remains a long-lasting challenge. To overcome this challenge, herein we present an acoustic differential bioassembly technique to assemble different cell types at the distinct positions of the acoustic field based on their inherent physical characteristics including cellular size and buoyant density. Specifically, different cell types can be differentially assembled beneath the nodal or the antinode regions of the Faraday wave to form complementary cytoarchitectures, or be selectively positioned at the center or edge area beneath either the nodal or the antinode regions to form sandwich cytoarchitectures. Using this technique, we assemble human induced pluripotent stem cell-derived liver spheroids and endothelial cells into hexagonal cytoarchitecturesin vitroto mimic the cord and sinusoid structures in the hepatic lobules. This hepatic lobule model reconstitutes liver metabolic and synthetic functions, such as albumin secretion and urea production. Overall, the acoustic differential bioassembly technique facilitates the construction of human relevantin vitroorganotypic models with spatially-defined heterocellular architectures, and can potentially find wide applications in tissue engineering and regenerative medicine.

Development of a high-throughput micropatterned agarose scaffold for consistent and reproducible hPSC-derived liver organoids.

Liver organoids represent emerging human-relevantin vitroliver models that have a wide range of biomedical applications in basic medical studies and preclinical drug discovery. However, the generation of liver organoids currently relies on the conventional Matrigel dome method, which lacks precise microenvironmental control over organoid growth and results in significant heterogeneity of the formed liver organoids. Here, we demonstrate a novel high-throughput culture method to generate uniform liver organoids from human pluripotent stem cell-derived foregut stem cells in micropatterned agarose scaffold. By using this approach, more than 8000 uniformly-sized liver organoids containing liver parenchyma cells, non-parenchymal cells, and a unique stem cell niche could be efficiently and reproducibly generated in a 48-well plate with a size coefficient of variation significance smaller than that in the Matrigel dome. Additionally, the liver organoids highly expressed liver-specific markers, including albumin (ALB), hepatocyte nuclear factor 4 alpha (HNF4α), and alpha-fetoprotein (AFP), and displayed liver functions, such as lipid accumulation, glycogen synthesis, ALB secretion, and urea synthesis. As a proof of concept, we evaluated the acute hepatotoxicity of acetaminophen (APAP) in these organoids and observed APAP-induced liver fibrosis. Overall, we expect that the liver organoids will facilitate wide biomedical applications in hepatotoxicity analysis and liver disease modeling.

Outcomes of Anti-CD19 CAR-T Treatment of Pediatric B-ALL with Bone Marrow and Extramedullary Relapse.

PURPOSE: Anti-CD19 chimeric antigen receptor T-cell immunotherapy (19CAR-T) has achieved impressive clinical results in adult and pediatric relapsed/refractory (r/r) B-lineage acute lymphoblastic leukemia (B-ALL). However, the application and effect of CAR-T therapy in B-ALL patients with extramedullary relapse are rarely issued even disqualified in some clinical trials. Here, we examined the efficacy of 19CAR-T in patients with both bone marrow and extramedullary involvement. MATERIALS AND METHODS: CAR-T cells were generated by transfection of primary human T lymphocytes with a lentiviral vector expressing anti-CD19 single chain antibody fragments (scFvs) with the cytoplasmic domains of 4-1BB and CD3ζ, and used to infuse patients diagnosed as having r/r B-ALL with extramedullary origination. Clinical responses were evaluated by the use of bone marrow aspiration, imaging, and flow cytometry. RESULTS: Eight patients received 19CAR-T infusion and all attained complete remission (CR). Only one patient was bridged to hematopoietic stem cell transplantation (HSCT). Although three patients relapsed after infusion, they received 19/22CAR-T infusion sequentially and attained a second remission. To date, five patients are in continuous CR and all eight patients are still alive. The mean follow-up time was 21.9 months, while the 24-month estimated event-free survival is 51.4%. CONCLUSION: 19CAR-T therapy can lead to clinical remission for extramedullary relapsed pediatric B-ALL patients. However, the problem of CD19+ relapses after CAR-T remained to be solved. For patients relapsing after CAR-T, a second CAR-T therapy creates another opportunity for remission for subsequent HSCT.

Asparagine synthetase is partially localized to the plasma membrane and upregulated by L-asparaginase in U937 cells.

This study investigated the intracellular localization of asparagine synthetase (ASNS) in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

[Correlation between blood T-cell receptor rearrangement excision circles levels and severe infection in children with acute lymphoblastic leukemia].

OBJECTIVE: This study quantitatively examined signal joint T-cell receptor rearrangement excision circles (sjTRECs) levels in peripheral blood of children with acute lymphoblastic leukemia (ALL) at different stages in order to evaluate the role of sjTRECs in predicting severe infection postchemotherapy. METHODS: sjTRECs levels in peripheral blood were measured by fluorescent quantitation-polymerase chain reaction in 30 children with newly diagnosed ALL, 36 children with ALL who accepted chemotherapy but were not infected, 30 children with ALL who had severe infection after chemotherapy, and 50 normal children. RESULTS: Blood sjTRECs levels in the normal group (394 ± 270 copies/103 MNC) were significantly higher than those in the other three groups (P<0.05). Blood sjTRECs levels in the chemotherapy group without infection (96 ± 78 copies/103 MNC) were significantly lower than those in the newly diagnosed ALL group (210 ± 219 copies/103 MNC) (P<0.05). The chemotherapy group with severe infection showed the lowest blood sjTRECs levels (48 ± 40 copies/103 MNC) in the four groups. CONCLUSIONS: The measurement of blood sjTRECs levels might be helpful for predicting the occurrence of severe infection postchemotherapy in children with ALL.

Outcome of Chinese children with unrelated donor hematopoietic stem cell transplantation.

BACKGROUND: To evaluate the prognostic factors and outcomes in Chinese children undergoing unrelated donor hematopoietic stem cell transplantation (UDT). METHODS: Retrospective analysis of clinical data from 53 consecutive children who received UDT from November 2002 to December 2007 in our center. RESULTS: The median recipient age was 8.4 years (range 1.5-21). With a median follow-up of 36 months (range 18-80), the probability of 3-year overall survival (OS) was 71.5%. Treatment-related mortality (TRM) was 19.0%, and 9.5% died after post-transplant leukemia relapse. Incidence of grades I-II, III-IV acute and chronic graft versus host disease (GVHD) was 63%, 29%, and 46%. There was significant difference in OS between patients older or younger than 10 years (50.0% vs. 84.8%, P = 0.003), patients with different underlying diseases (ALL, AML, CML, and non-malignant disease: 36.4%, 80.0%, 61.5%, and 100%, P = 0.001) and patients receiving either HLA 0-1 versus 2-3 loci high-resolution mismatched UDT (83.3% vs. 53.3%, P = 0.034). The OS was not affected by the stem cell source (peripheral stem cell 70.3%, bone marrow 87.5% vs. cord blood 62.5%, P = 0.542) or the severity of acute GVHD (grade 0-II 77.8% vs. grade III-IV 60.0%, P = 0.140). CONCLUSIONS: The important prognostic factors for OS after UDT were the degree of HLA match, the age of patient and the type of underlying disease. Patients < 10-year with non-malignant disease receiving 0-1 locus high-resolution mismatched UDT had the most favorable outcomes.