Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Model-Informed Precision Dosing of Intravenous Busulfan in Thai Pediatrics Undergoing Hematopoietic Stem Cell Transplantation.

BACKGROUND: Conditioning bifunctional agent, busulfan, is commonly used on children before hematopoietic stem cell transplantation. Currently, at the Ramathibodi hospital, Bangkok, Thailand, initial dosing is calculated according to age and body surface area, and 7 samples per day are used for therapeutic drug monitoring (TDM). This study aimed to identify the best strategies for individual dosages a priori from patient characteristics and a posteriori based on TDM. METHODS: The pharmacokinetic data set consisted of 2018 plasma concentrations measured in 135 Thai (n = 135) pediatric patients (median age = 8 years) and were analyzed using a population approach. RESULTS: Body weight, presence of malignant disease, and genetic polymorphism of Glutathione S-transferase Alpha-1 (GSTA1) were predictors of clearance. The optimum sampling times for TDM concentration measurements were 0.25, 2, and 5 hours after a 3-hour infusion. This was sufficient to obtain a Bayesian estimate of clearance a posteriori. Simulations showed the poor performance of a priori formula-based dose calculations with 90% of patients demonstrating a 69%-151% exposure interval around the target. This interval shrank to 85%-124% if TDM was carried out only at day 1 and to 90%-116% with TDM at days 1 and 3. CONCLUSIONS: This comprehensive study reinforces the interest of TDM in managing interindividual variability in busulfan exposure. Therapeutic drug monitoring can reliably be implemented from 3 samples using the Bayesian approach, preferably over 2 days. If using the latter is not possible, the formulas developed herein could present an alternative in Thai patients.

Nationwide Study of Factors Impacting Survival Outcome and Consequences in Children with Reactivation/Refractory Langerhans Cell Histiocytosis.

BACKGROUND: Disease reactivation/refractory remains a major challenge in managing Langerhans cell histiocytosis (LCH). Outcomes and late sequelae should be explored. METHODS: A multi-institutional retrospective study was conducted to describe clinical characteristics, predictive factors, outcomes and late sequelae of pediatric reactivation/refractory LCH in Thailand. RESULTS: In all, 47 patients were studied, 25 (53.2%) patients had disease reactivation and 22 (46.8%) patients had refractory LCH. The median reactivation and refractory time were 1.59 and 0.33 years from diagnosis, respectively (p <0.001). The most common site of reactivation/refractory was the bone (n = 26, 55%), and 20 (42.6%) patients developed late sequelae. The 5-year overall survival (OS) was 76.1%. Patients with reactivation and refractory LCH performed similarly in 5-year OS (88% vs. 63%, p = 0.055). Prognostic factors associated with mortality were liver, spleen, hematopoietic system and lung reactivation (p <0.05). Lung reactivation was the only independent risk factor associated with the survival outcome (p = 0.002). CONCLUSIONS: The outcomes of pediatric patients between reactivation and refractory LCH in Thailand were similarly desirable and mortality was minimal although late sequelae may evolve. Pulmonary reactivation/refractory was an independent risk factor associated with survival.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients.

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

Immune reconstitution in children after haploidentical haematopoietic stem cell transplantation.

INTRODUCTION: Immune reconstitution (IR) kinetics of paediatric patients underwent haploidentical haematopoietic stem cell transplantation (HSCT) with post-transplant cyclophosphamide (PTCy) have not been extensively studied. We compared IR patterns of children receiving HSCT from haploidentical (n = 92) and HLA-matched donors (n = 36), and analysed risk factors for viral infection in these patients. METHODS: We prospectively measured lymphocyte subset numbers before HSCT and at 1, 3, 6 and 12 months after HSCT. Blood cytomegalovirus (CMV), Epstein-Barr virus, adenovirus, BK virus (BKV) and urine adenovirus and BKV viral loads were measured at designated time points. RESULTS: The median numbers of total T and T helper cells at 1 month were significantly lower in the haploidentical group compared with the HLA-matched group. Haploidentical HSCT recipients had significantly lower median numbers of several T cell subsets and B cells for 1 year after HSCT. The median NK cell count of the haploidentical group was lower at 1 month. BKV haemorrhagic cystitis, blood CMV and urine adenovirus reactivation were more frequently found in the haploidentical group. Post-haploidentical HSCT patients receiving anti-T lymphocyte globulin (ATG) had significantly lower median numbers of total T cells (at 1 month) and T helper cells (at 6 and 12 months) and higher rate of blood BKV reactivation compared with those without ATG. CONCLUSION: Paediatric patients who undergo haploidentical HSCT with PTCy are likely to have delayed IR and an increased risk of viral reactivation/infection compared with HLA-matched HSCT. The addition of ATG to PTCy delayed T cell recovery and increased risk of BKV reactivation.

Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative.

BACKGROUND: The diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown. METHOD: We utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis. RESULT: The supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73- hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs. CONCLUSION: These findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells.

BACKGROUND AIMS: Gene therapy using lentiviral vectors (LVs) that harbor a functional ß-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). METHODS: After HSPCs were transduced with an LV encoding the therapeutic ß-globin (ßA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. RESULTS: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of ßA-T87Q protein detected by reverse-phase high-performance liquid chromatography. CONCLUSIONS: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

HIV-1 proviral DNA in purified peripheral blood CD34+ stem and progenitor cells in individuals with long-term HAART; paving the way to HIV gene therapy.

Human immunodeficiency virus (HIV)-1 infection is an important public health problem worldwide. After primary HIV-1 infection, transcribed HIV-1 DNA is integrated into the host genome, serving as a reservoir of the virus and hindering a definite cure. Although highly active antiretroviral therapy suppresses active viral replication, resulting in undetectable levels of HIV RNA in the blood, a viral rebound can be detected after a few weeks of treatment interruption. This supports the concept that there is a stable HIV-1 reservoir in people living with HIV-1. Recently, a few individuals with HIV infection were reported to be probably cured by hematopoietic stem transplantation (HSCT). The underlying mechanism for this success involved transfusion of uninfected hematopoietic stem and progenitor cells (HSPCs) from CCR5-mutated donors who were naturally resistant to HIV infection. Thus, gene editing technology to provide HIV-resistant HSPC has promise in the treatment of HIV infections by HSCT. In this study, we aimed to find HIV-infected individuals likely to achieve a definite cure via gene editing HSCT. We screened for total HIV proviral DNA by Alu PCR in peripheral blood mononuclear cells (PBMCs) of 20 HIV-infected individuals with prolonged viral suppression. We assessed the amount of intact proviral DNA via a modified intact proviral DNA assay (IPDA) in purified peripheral CD34+ HSPCs. PBMCs from all 20 individuals were positive for the gag gene in Alu PCR, and peripheral CD34+ HSPCs were IPDA-negative for six individuals. Our results suggested that these six HIV-infected individuals could be candidates for further studies into the ability of gene editing HSCT to lead to a definite HIV cure.

Ceftriaxone exerts antitumor effects in MYCN-driven retinoblastoma and neuroblastoma by targeting DDX3X for translation repression.

MYCN proto-oncogene, bHLH transcription factor (MYCN) amplification is associated with aggressive retinoblastoma (RB) and neuroblastoma (NB) cancer recurrence that is resistant to chemotherapies. Therefore, there is an urgent need to identify new therapeutic tools. This study aimed to evaluate the potential repurposing of ceftriaxone for the treatment of MYCN-amplified RB and NB, based on the clinical observations that the drug was serendipitously found to decrease the volume of the MYCN-driven RB subtype. Using patient-derived tumor organoids and tumor cell lines, we demonstrated that ceftriaxone is a potent and selective growth inhibitor targeting MYCN-driven RB and NB cells. Profiling of drug-induced transcriptomic changes, cell-cycle progression, and apoptotic death indicated cell-cycle arrest and death of drug-treated MYCN-amplified tumor cells. Drug target identification, using an affinity-based proteomic and molecular docking approach, and functional studies of the target proteins revealed that ceftriaxone targeted DEAD-box helicase 3 X-linked (DDX3X), thereby inhibiting translation in MYCN-amplified tumors but not in MYCN-nonamplified cells. The data suggest the feasibility of repurposing ceftriaxone as an anticancer drug and provide insights into the mechanism of drug action, highlighting DDX3X as a potential target for treating MYCN-driven tumors.

Cytotoxicity and exhaustion markers of chimeric antigen receptor T cells targeting BCMA in multiple myeloma cell lines between patients and healthy donors.

OBJECTIVES: Multiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform. METHODS: BCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA-ECTM and K562, respectively. RESULTS: BCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA-ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM-3, LAG-3, and PD1. CONCLUSIONS: Our BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA-expressing cells in vitro and had similar levels of exhaustion markers among different populations.

Screening for ELANE, HAX1 and GFI1 gene mutations in children with neutropenia and clinical characterization of two novel mutations in ELANE gene.

BACKGROUND: Congenital neutropenia is a rare disease. Recurrent infections since young age are the presentation. The most common mutation causing severe congenital neutropenia (SCN) and cyclic neutropenia (CyN) is the ELANE gene. The objectives of this study were to screen the three common genetic mutations of ELANE, HAX1 and GFI1 in children with chronic neutropenia and to describe the clinical characteristics of children who had the mutations. METHODS: Infants having ANC < 1,000/cu mm or children aged > 1 year having ANC < 1,500/cu mm at least 3 times in 3 months were enrolled in the study. Patients who had acquired neutropenia due to infection, immune deficiency, or drugs were excluded. The ELANE gene was first studied; and if mutations were not identified, the HAX1 and GFI1 genes were further examined. RESULTS: A total of 60 patients were enrolled in the study. The median (range) age, ratio of female to male, ANC, and last follow-up age were 9.2 (0.5-45.2) months, 1:1.2, 248 (0-1,101) /cu mm, and 19.9 (3.5-202.3) months, respectively. Infections were noted in 67.3% of all patients. ELANE gene mutation was found in only four patients (6.7%), and the rest (56 patients) showed no mutations in the HAX1 and GFI1 genes. In patients without mutations, 66.0% had normal ANC during the follow-up, with a median (range) age for normal ANC of 19.8 (4.0-60.0) months. Two novel mutations p. Ala79del (c.234_236del) and p. Val197GlufsTer18 (c.589_590insAGGCCGGC) were identified, and they respectively cause SCN and CyN. Patients with the two novel mutations presented with several episodes of infection, including pneumonia, sepsis, abscess, otitis media, and gum infection. CONCLUSION: The genetic screening for ELANE, HAX1, and GFI1 gene mutations in 60 patients with chronic neutropenia could identify four patients (6.7%) with ELANE gene mutation and two novel mutations, p. Ala79del in exon 3 and p. Val197GlufsTer18 in exon 4 causing SCN; and CyN, respectively.

Cytomegalovirus-Specific T Cells in Pediatric Liver Transplant Recipients.

Cytomegalovirus (CMV) infection is a major opportunistic infection after liver transplantation (LT) that necessitates monitoring. Because of the lack of studies in children, we aimed to investigate CMV-specific T cell immune reconstitution among pediatric LT recipients. The recipients were monitored for CMV infection and CMV-specific T cells from the start of immunosuppressive therapy until 48 weeks after LT. Clinically significant CMV viremia (csCMV) requiring preemptive therapy was defined as a CMV load of >2000 IU/mL. Peripheral blood CMV-specific T cells were analyzed by flow cytometry based on IFNγ secretion upon stimulation with CMV antigens including immediate early protein 1 (IE1) Ag, phosphoprotein 65 (pp65) Ag, and whole CMV lysate (wCMV). Of the 41 patients who underwent LT, 20 (48.8%) had csCMV. Most (17/20 patients) were asymptomatic and characterized as experiencing CMV reactivation. The onset of csCMV occurred approximately 7 weeks after LT (interquartile range: 4-12.9); csCMV rarely recurred after preemptive therapy. Lower pp65-specific CD8+ T cell response was associated with the occurrence of csCMV (p = 0.01) and correlated with increased viral load at the time of csCMV diagnosis (ρ = -0.553, p = 0.02). Moreover, those with csCMV had lower percentages of IE1-specific CD4+ and wCMV-reactive CD4+ T cells at 12 weeks after LT (p = 0.03 and p = 0.01, respectively). Despite intense immunosuppressive therapy, CMV-specific T cell immune reconstitution occurred in pediatric patients post-LT, which could confer protection against CMV reactivation.

Establishment of MUi030-A: A human induced pluripotent stem cell line carrying homozygous L444P mutation in the GBA1 gene to study type-3 Gaucher disease.

Gaucher disease (GD) is a common lysosomal storage disease resulting from mutations in the glucocerebrosidase (GBA1) gene. This genetic disorder manifests with symptoms affecting multiple organs, yet the underlying mechanisms leading to pathology remain elusive. In this study, we successfully generated the MUi030-A human induced pluripotent stem cell (hiPSC) line using a non-integration method from a male type-3 GD patient with a homozygous c.1448T>C (L444P) mutation. These hiPSCs displayed a normal karyotype and pluripotency markers and the remarkable ability to differentiate into cells representing all three germ layers. This resourceful model holds significant promise for illuminating GD's underlying pathogenesis.

PCR-based versus conventional stool tests in children with diarrhea who underwent solid organ transplantation or hematopoietic stem cell transplantation.

Infectious diarrhea is a common problem among post-transplant recipients. Compared to conventional tests, polymerase chain reaction (PCR)-based stool tests have been shown to improve diagnostic yield but the aforementioned data in children remain limited. Our aims were to assess the detection rate of PCR-based tests in post-transplant children and compare with the conventional tests; and to investigate how these stool tests help in managing these children. We enrolled children aged 1 to 19 years who underwent solid organ transplantation or hematopoietic stem cell transplantation that remained on immunosuppressive agents and developed diarrhea ≥ 24 hours between January 2015 and February 2023. Besides stool tests, data on demographics, clinical characteristics and management were collected. We analyzed 68 patients and 92 episodes of diarrhea with PCR-based tests. PCR-based tests provided a detection rate of 41.8% versus 16.5% for the conventional tests. While conventional tests may detect a higher proportion of Clostridiodes difficile infection, PCR-based tests showed greater yields in detecting Salmonella spp. and viruses especially norovirus. PCR-based tests had an impact in management among 22/38 (58%) diarrheal episodes especially with Campylobacter jejuni and C difficile; and among 16 episodes that positive PCR-based tests had a minimal impact, the most common reason was due to the need for continuation of antimicrobial agents for concomitant site-specific infection (69%). Among transplanted children presenting with diarrhea, PCR-based tests provide a higher yield when compared with the conventional tests. The PCR-based stool tests may also further guide clinicians for providing proper antimicrobial agents.

A novel anti-membrane CD30 single-chain variable fragment discovered from the human phage library: A potential targeted immunotherapy.

Hodgkin's lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers.

Genomics-Driven Precision Medicine in Pediatric Solid Tumors.

Over the past decades, several study programs have conducted genetic testing in cancer patients to identify potential genetic targets for the development of precision therapeutic strategies. These biomarker-driven trials have demonstrated improved clinical outcomes and progression-free survival rates in various types of cancers, especially for adult malignancies. However, similar progress in pediatric cancers has been slow due to their distinguished mutation profiles compared to adults and the low frequency of recurrent genomic alterations. Recently, increased efforts to develop precision medicine for childhood malignancies have led to the identification of genomic alterations and transcriptomic profiles of pediatric patients which presents promising opportunities to study rare and difficult-to-access neoplasms. This review summarizes the current state of known and potential genetic markers for pediatric solid tumors and provides perspectives on precise therapeutic strategies that warrant further investigations.

Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models.

T cells genetically engineered to express a chimeric antigen receptor (CAR) specifically binding to a CD19 antigen has become the frontline of hematological malignancies immunotherapy. Their remarkable antitumor effect has exerted complete remission in treating B-cell malignancies. Although successful patient treatment has been shown, improvement to the structure of CAR to enhance its safety and efficacy profile is warranted. Transduction with a lentiviral vector (LVV) leading to the expression of CARs is also a critical step in redirecting T cells to target specific tumor antigens. To improve the efficacy of CD19 CARs in this study, the transduction ability of second and third generations LVV were compared. Ex vivo expansion of CD19 CARs T cells from healthy donors' peripheral blood mononuclear cells was performed after transduction of T cells with second and third generations LVV. Transduction efficacy of transduced T cells was determined to show a higher percentage in the third generations LVV transduced cells, with no changes in viability and identity of cells characterized by immunophenotyping. Testing the cytotoxic capacity of third generations LVV-transduced T cells against target cells showed higher reactivity against control cells. Cytokine expression was detected on the CD19 CARs T cells, suggesting that these cells limit in vitro growth of B-cell leukemia via secretion of the pro-inflammatory cytokine IFN γ. To investigate whether the third generation LVV transduced T cells can limit CD19 lymphoma growth in vivo, an analysis of tumor burden in a mouse model assessed by bioluminescence imaging was performed. We found that, in the presence of CD19 CARs T cells, the level of tumor burden was markedly reduced. In addition, an increase in the length of survival in mice receiving CAR-CD19 T cells was also observed. This suggests that transduction with third generations LVV generate a functional CAR-CD19 T cells, which may provide a safer and effective therapy for B-cell malignancies.

Effective T-cell replete haploidentical stem cell transplantation for pediatric patients with high-risk hematologic disorders.

OBJECTIVES: Patients with high-risk hematologic diseases require intensive modalities, including high-dose chemotherapy and allogeneic hematopoietic stem cell transplantation (allo-HSCT). Haploidentical T-cell-replete transplantation is a logical choice because of the limited availability of matched sibling donors and the prolonged time needed to identify matched unrelated donors in Thailand. METHODS: The clinical outcomes data of 43 patients undergoing allo-HSCT were reviewed. All patients had high-risk hematologic malignancies, were younger than 20 years, and were in complete cytological remission at the time of allo-HSCT. We used two different conditioning regimens: total body irradiation (TBI) combined with cyclophosphamide, fludarabine, and melphalan (n = 23) and thiotepa combined with fludarabine and busulfan (n = 20). All patients received a graft-versus-host disease prophylaxis regimen consisting of cyclophosphamide, mycophenolate mofetil, and a calcineurin inhibitor or sirolimus. RESULTS: There was no difference in engraftment between patients receiving either of the regimens. After a median follow-up of 35.8 (range, 0.6-106.2) months, the overall survival (OS) and event-free survival (EFS) rates were 62.4% and 54.7%, respectively. OS and EFS were comparable between the respective regimens. CONCLUSIONS: We conclude that thiotepa-based conditioning has similar efficacy and tolerability as TBI-based conditioning for haploidentical HSCT with post-transplant cyclophosphamide.

Comprehensive and long-term outcomes of enzyme replacement therapy followed by stem cell transplantation in children with Gaucher disease type 1 and 3.

BACKGROUND: Gaucher disease (GD) is a lysosomal storage disorder, characterized by hepatosplenomegaly, pancytopenia, bone diseases, with or without neurological symptoms. Plasma glucosylsphingosine (lyso-Gb1), a highly sensitive and specific biomarker for GD, has been used for diagnosis and monitoring the response to treatment. Enzyme replacement therapy (ERT) is an effective treatment for the non-neurologic symptoms of GD. Neuronopathic GD (type 2 and 3) accounts for 60%-70% of the Asian affected population. METHODS: We explored combination therapy of ERT followed by hematopoietic stem cell transplantation (HSCT) and its long-term outcomes in patients with GD type 3 (GD3). RESULTS: Four patients with GD3 and one with GD type 1 (GD1) underwent HSCT. The types of donor were one matched-related, one matched-unrelated, and three haploidentical. The age at disease onset was 6-18 months and the age at HSCT was 3.8-15 years in the patients with GD3. The latest age at follow-up was 8-22 years, with a post-HSCT duration of 3-14 years. All patients had successful HSCT. Chronic graft-versus-host disease occurred in one patient. The enzyme activities were normalized at 2 weeks post HSCT. Lyso-Gb1 concentrations became lower than the pathological value. All of the patients are still alive and physically independent. Most of them (4/5) returned to school. None of the patients with GD3 had seizures or additional neurological symptoms after HSCT, but showed varying degrees of cognitive impairment. CONCLUSIONS: ERT followed by HSCT could be considered as an alternative treatment for patients with GD3 who have a high risk of fatal neurological progression.