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The prognosis of over 90% of infant acute lymphoblastic leukemia (ALL) remains poor because of harboring the mixed-lineage leukemia gene (MLL) fusion. To give insight into the critical coexpressed genes related to the MLL-rearrangement (MLL-R) gene in childhood acute lymphoblastic leukemia, we integrated different bioinformatic methods. First, the gene expression data of MLL-R ALL and normal samples from GSE13159 and GSE13164 were analyzed using “compare” function in the Oncomine database. The top 150 overexpressed and 150 underexpressed genes were identified by the Oncomine website. Then, we employed the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) to define functional genes for the 300 DEGs. The Cytoscape identified two important networks for overexpressed genes, including 35 functional genes, among which PROM1, FLT3, CTGF, LGALS1, IGFBP7, ZNRF1, and RUNX2 were considered as the key genes because of their high expression in MLL-R ALL compared to the expression in other subclassification of leukemia in the MILE dataset. Further analysis of GSE68720, GSE19475, and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ALL (phase I) database confirmed the robust expression of 7 key genes in MLL-R compared to MLL-germline (MLL-G) childhood ALL. Kaplan-Meier analysis indicated that childhood ALL patients with high PROM1 and CTGF expression had significantly poor overall survival. These findings suggest that PROM1 and CTGF represent two potential therapeutic targets for childhood MLL-R ALL.

Haematopoietic cell transplantation (HCT) is the only curative treatment for haematological complications in patients with Fanconi anaemia (FA). Haploidentical (haplo-) HCT is a promising alternative for FA. We aimed to analyse the outcomes of unmanipulated haplo-HCT in patients with FA with radiation-free conditioning. A total of 56 patients from 11 centres between 2013 and 2021 in China were retrospectively analysed. The mean (SD) cumulative incidence was 96.4% (0.08%) for 30-day neutrophil engraftment and 85.5% (0.24%) for 100-day platelet engraftment. With a median (range) follow-up of 2.4 (0.2-5.8) years, favourable mean (SD) overall survival of 80.9% (5.5%) and event-free survival of 79.3% (5.6%) were achieved. The mean (SD) incidences of acute graft-versus-host disease (aGvHD) Grade II-IV and Grade III-IV were 55.4% (0.45%) and 42.9 (0.45%) respectively. The mean (SD) cumulative incidence of 3-year chronic graft-versus-host disease (cGvHD) was 34.7% (0.86%) and that of moderate-to-severe cGvHD was 9.0% (0.19%). Our data demonstrate that in unmanipulated haplo-HCT for patients with FA, radiation-free regimens based on fludarabine and low-dose cyclophosphamide ± busulfan achieved favourable engraftment and survival with relatively high incidences of aGvHD and cGvHD. These results prompt the use of low-intensity conditioning without radiation and intensive GvHD prophylaxis when considering unmanipulated haplo-HCT in patients with FA.

IntroductionThe emergence of multidrug-resistant Pseudomonas aeruginosa poses a global threat, but the distribution and resistance profiling are unclear, especially in young children. Infections due to P. aeruginosa are common, associated with high mortality, and increasingly β-lactam drug resistant.MethodsWe studied the molecular epidemiology and antibiotic resistance mechanisms in 294 clinicalisolates of P. aeruginosa from a pediatric hospital in China. Non-duplicate isolates were recovered from clinical cases and were identified using an API-20 kit followed by antimicrobial susceptibility testing using the VITEK®2 compact system (BioMerieux, France) and also by broth dilution method. In addition, a double-disc synergy test for the ESBL/E-test for MBL was performed. The presence of beta-lactamases, plasmid types, and sequence types was determined by PCR and sequencing.ResultsFifty-six percent (n = 164) of the isolates were resistant to piperacillin–tazobactam, followed by cefepime (40%; n = 117), ceftazidime (39%; n = 115), imipenem (36%; n = 106), meropenem (33%; n = 97), and ciprofloxacin (32%; n = 94). Forty-two percent (n = 126) of the isolates were positive for ESBL according to the double-disc synergy test. The blaCTX-M-15 cephalosporinase was observed in 32% (n = 40/126), while 26% (n = 33/126) werepositive for blaNDM-1 carbapenemase. Aminoglycoside resistance gene aac(3)IIIawas observed in 16% (n = 20/126), and glycylcyclines resistance gene tet(A) was observed in 12% (n = 15/126) of the isolates. A total of 23 sequence types were detected, including ST1963 (12%; n = 16), followed by ST381 (11%; n = 14), ST234 (10%; n = 13), ST145 (58%; n = 10), ST304 (57%; n = 9), ST663 (5%; n = 7), and a novel strain. In ESBL-producing P. aeruginosa, 12 different Incompatibility groups (Inc) were observed, the most common being IncFI, IncFIS, and IncA/C. The MOBP was the most common plasmid type, followed by MOBH, MOBF, and MOBQ.DiscussionOur data suggest that the spread of antibiotic resistance is likely due toclonal spread and dissemination of different clinical strains of P. aeruginosa harbouring different plasmids. This is a growing threat in hospitals particularly in young children which needs robust prevention strategies.

A 4-year-old child with β-thalassemia major underwent allogeneic hematopoietic stem cell transplantation (HSCT) on August 30, 2022 from a male haploidentical donor. He developed massive pericardial effusion (PE) secondary to a pulmonary infection. Methylprednisolone, tacrolimus discontinued, and pericardiocentesis were all ineffective. Since the interleukin-6 (IL-6) level in the PE was markedly increased (1868.58pg/ml), a total of two doses of tocilizumab was administered intravenously at a dosage of 5mg/kg every 2 weeks. PE remarkably reduced and completely resolved within 8 days. No recurrence of PE was observed in the follow up visits. Our results suggest that tocilizumab can be considered as a treatment of choice in massive PE after HSCT.

BackgroundGene therapy is gradually becoming recognized as a possibly curative therapeutic strategy for transfusion-dependent β-thalassemia (TDT). Gene therapy addresses the problem of donor scarcity through the application of autologous hematopoietic stem cells (HSCs), which also can reduce the risks that accompany allogeneic HSC transplantation. When using gene addition strategy, lentiviral vector is critical for the efficacy and safety of β-thalassemia gene therapy. In our preclinical studies, LentiHBBT87Qvector with optimized backbone was developed to efficiently restore β–globin expression in HSCs-derived erythroblasts of TDT patients with minimal risk of tumorigenesis. Here, we presented the clinical trial results of gene therapy using LentiHBBT87Qvector in two TDT patients.MethodIn an ongoing phase 1/2 trial (NCT05745532), auto-HSCs were mobilized from two TDT patients, and then transduced with LentiHBBT87Qvector. The gene-modified auto-HSCs is called HGI-001 injection. After four-day consecutive myeloablative conditioning, these two patients were administrated with HGI-001 injection via intravenous infusion. Medical examinations were performed in the transplantation unit to monitor patients’ status till the patients were clinically stable. Then, 24-month following-up visits are conducted to assess the safety and efficacy of HGI-001 injection. The safety endpoints of this clinical study include the incident and severity of adverse events (AEs); transplant-related mortality or disability events within 100 days post drug product infusion; vector-related replication competent lentivirus (RCL) and clonal variations containing specific viral integration sites; overall survival during this clinical trial. The major efficacy endpoint is the percentage of subjects with average vector copy number (VCN) in peripheral blood mononuclear cells (PBMCs) >0.1, and average expression of exogenous HbAT87Q>2.0g/dL at the 24thmonth after reinfusion of HGI-001 injectionResultsThe rapid neutrophil and platelet engraftment successfully happened after reinfusion of HGI-001 injection. The two patients with non-β0/β0genotype have been transfusion-independent for 24 months and 21 months post-treatment. At the last visit, the levels of HbAT87Qare 7.3 and 6.9g/dL, and the levels of total hemoglobin are 9.8 and 10.1 g/dL. After the two subjects stopped transfusions, the iron overload has been alleviated without iron chelation treatment. Most AEs are myeloablative conditioning related, and can be controlled through clinically standard therapeutic managements. No clone dominance related to vector integration nor RCL has been observed.ConclusionGene therapy with optimized LentiHBBT87Qvector (HGI-001 injection) assist two TDT patients become transfusion-independent without serious adverse events related to the product.

Background Blinatumomab could be successfully used to reduce minimal residual disease (MRD) prior to hematopoietic stem cell transplantation (HSCT) in pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), but sound evidence is lacking in China. Case presentation This retrospective study assessed the application of blinatumomab in B-ALL accompanied by persistent or relapsed low-level MRD before HSCT from April 2019 to July 2021. Two cases (Cases 1 and 2) initially achieved remission with MRD Conclusions Blinatumomab therapy showed a good performance for three pediatric cases with detectable but low MRD before HSCT in China. However, further prospective studies with large sample sizes are still needed for further clarification.

Background Antimicrobial resistance is a global challenging issue in children and adults. Finding the extent of resistance is the first step in finding an appropriate way to combat it. This study aimed to assess the antibiotic resistance patterns of different bacterial isolates in urine specimens from children and adults. Methods This retrospective cross-sectional study was done from January 1, 2014, to December 30, 2018, and used data from bacterial cultures collected and processed at the Centre Universitaire de Diagnostic au Graben (CUDG), located in Butembo in the Eastern part of the Democratic Republic of Congo. Conventional standard urine culture followed by bacteria identification based on conventional methods (colony morphology, Gram stain, and biochemical tests) were performed. Standard disc diffusion drug susceptibility testing was performed using CLSI M 100-S22 guidelines. Findings Of 1620 urine specimens collected, 1041 (64.3%) showed positive microorganism growth. The three most isolated bacterial microorganisms were S. aureus (561 [53.9%]), E. coli (124 [11.9%]), and Streptococcus spp (74 [7.1%]). Resistance of S. aureus was as follows: amoxicillin/clavulanic acid 50.3% (234 of 465 isolated tested), gentamicin 37.9% (167/441), ceftriaxone (329/469), ciprofloxacin (207/509), doxycycline (232/524), nalidixic acid (73/92) and meropenem (64/88). The resistance profile of E. coli was as follows: amoxicillin/clavulanic acid 77.8% (70/90), gentamicin % (44/95), ceftriaxone (45/108), ciprofloxacin (55/113), doxycycline (78/108), nalidixic acid (56/73) and meropenem (33/55). Resistance of Streptococci spp was amoxicillin/clavulanic acid 34.9% (23/66), gentamicin % (26/56), ceftriaxone (28/62), ciprofloxacin (28/66), doxycycline (28/64), nalidixic acid (14/17) and meropenem (7/8). Among the isolated bacteria, 66.8% (695/1041) were multidrug-resistant. Conclusion Antimicrobial resistance is prevalent among isolated uropathogenic bacteria, especially to first-line and second-line antibiotics. Continued surveillance and a tracking system for multidrug-resistant bacteria are needed. Judicious and rational antibiotic usage is recommended.

Long noncoding ribonucleic acids (RNAs; LncRNAs) endowed with both protein-coding and noncoding functions are referred to as ‘dual functional lncRNAs’. Recently, dual functional lncRNAs have been intensively studied and identified as involved in various fundamental cellular processes. However, apart from time-consuming and cell-type-specific experiments, there is virtually no in silico method for predicting the identity of dual functional lncRNAs. Here, we developed a deep-learning model with a multi-head self-attention mechanism, LncReader, to identify dual functional lncRNAs. Our data demonstrated that LncReader showed multiple advantages compared to various classical machine learning methods using benchmark datasets from our previously reported cncRNAdb project. Moreover, to obtain independent in-house datasets for robust testing, mass spectrometry proteomics combined with RNA-seq and Ribo-seq were applied in four leukaemia cell lines, which further confirmed that LncReader achieved the best performance compared to other tools. Therefore, LncReader provides an accurate and practical tool that enables fast dual functional lncRNA identification.

Hematopoietic stem cells (HSCs) provide a life-long supply of haemopoietic cells and are indispensable for clinical transplantation in the treatment of malignant hematological diseases. Clinical applications require vast quantities of HSCs with maintained stemness characteristics. Meeting this demand poses often insurmountable challenges for traditional culture methods. Creating a supportive artificial microenvironment for the culture of HSCs, which allows the expansion of the cells while maintaining their stemness, is becoming a new solution for the provision of these rare multipotent HSCs. Hydrogels with good biocompatibility, excellent hydrophilicity, tunable biochemical and biophysical properties have been applied in mimicking the hematopoietic niche for the efficient expansion of HSCs. This review focuses on recent progress in the use of hydrogels in this specialized application. Advanced biomimetic strategies use for the creation of an artificial haemopoietic niche are discussed, advances in combined use of hydrogel matrices and microfluidics, including the emerging organ-on-a-chip technology, are summarized. We also provide a brief description of novel stimulus-responsive hydrogels that are used to establish an intelligent dynamic cell microenvironment. Finally, current challenges and future perspectives of engineering hydrogels for HSC biomedicine are explored.

Background The emergence of multi-drug-resistant Pseudomonas aeruginosa poses a global threat worldwide. We studied the molecular epidemiology and antibiotic resistance mechanisms in 294 clinical isolates of P. aeruginosa from a paediatric hospital. Methods Isolates were identified using an API-20 kit and antimicrobial susceptibility testing was performed using the VITEK®2 compact. β-lactamases, plasmid types and sequence types were determined by PCR and sequencing. The double-disc diffusion method for ESBL was performed. Results 56% (n = 164) isolates were resistant to piperacillin-tazobactam followed by cefepime 40% (n = 117), ceftazidime 39% (n = 115), imipenem 36% (n = 106), meropenem 33% (n = 97), ciprofloxacin 32% (n = 94). 42% (n = 126) isolates were positive for ESBL by double-disc diffusion. The blaCTX-M-15 cephalosporinase was observed in 32% (n = 40/126) while 26% (n = 33/126) were positive for blaNDM-1 carbapenemase. Aminoglycoside resistance gene aac(3)IIIa was observed in 16% (n = 20/126) and glycylcycline resistance gene tetA(Aa) in 12% (n = 15/126) of the isolates. A total of 23 sequence types were detected, ST1963, 12% (n = 16) followed by ST381, 11% (n = 14); ST234, 10% (n = 13; ST145, 58% (n = 10); ST304, 57% (n = 9); ST663 5% (n = 7) and a novel strain. In ESBL-producing P. aeruginosa, 12 different Incompatibility groups (Inc) were observed, the most common being IncFI, IncFIS and IncA/C. The MOBP was the most common plasmid type followed by MOBH, MOBF and MOBQ. Conclusion The spread of antibiotic resistance is likely due to clonal spread and circulation of different plasmids in clinical strains of P. aeruginosa.

Background: Next-generation sequencing (NGS) is an emerging technology that can comprehensively assess the diversity of the immune system. We explored the feasibility of NGS in detecting minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) based on immunoglobulin and T cell receptor.Methods: Bone marrow samples were collected pre- and post-treatment with pediatric ALL admitted to Shenzhen Children's Hospital from February 1st, 2020 to January 31st, 2021. We analyzed the MRD detected by NGS, multiparametric flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR), and analyzed risk factors of positive NGS-MRD at the end of B-ALL induction chemotherapy.Results: A total of paired 236 bone marrow samples were collected from 64 children with ALL (58 B-ALL and 6 T-ALL). The decrease in the clonal rearrangement frequency of IGH, IGK, and IGL was generally consistent after treatment. Positive MRD was detected in 57.5% (77/134) of B-ALL and 80% (12/15) of T-ALL by NGS after chemotherapy, which was higher than those detected by MFC and RQ-PCR. In B-ALL patients, MRD results detected by NGS were consistent with MFC（r = 0.708, p < 0.001）and RQ-PCR（r = 0.618, p < 0.001）. At the end of induction, NGS-MRD of 40.4% B-ALL was >0.01% and multivariate analysis indicated that ≧2 clonal rearrangement sequences before treatment were an independent factor of negative NGS-MRD. Conclusions: NGS is more sensitive than MFC and RQ-PCR for MRD measurement. B-ALL children with ≧2 clonal rearrangements detected by NGS before treatment are difficult to switch to negative MRD after chemotherapy.

The bioprosthetic heart valves (BHVs) are the best option for the treatment of valvular heart disease. Glutaraldehyde (Glut) is commonly used as the golden standard reagent for the crosslinking of BHVs. However, the obvious defects of Glut, including residual aldehyde toxicity, degradation and calcification, increase the probability of valve failure in vivo and motivated the exploration of alternatives. Thus, the aim of this study is to develop a non-glutaraldehyde hybrid cross-linking method composed of Neomycin Trisulfate, Polyethylene glycol diglycidyl ether and Tannic acid as a substitute for Glut, which was proven to reduce calcification, degradation, inflammation of the biomaterial. Evaluations of the crosslinked bovine pericardial included histological and ultrastructural characterization, biomechanical performance, biocompatibility and structural stability test, and in vivo anti-inflammation and anti-calcification assay by subcutaneous implantation in juvenile Sprague Dawley rats. The results revealed that the hybrid crosslinked bovine pericardial were superior to Glut crosslinked biomaterial in terms of better hydrophilicity, thermodynamics stability, hemocompatibility and cytocompatibility, higher Young’s Modulus, better stability and resistance to enzymatic hydrolysis, and lower inflammation, degradation and calcification levels in subcutaneous implants. Considering all above performances, it indicates that the hybrid cross-linking method is appropriate to replace Glut as the method for BHV preparation, and particularly this hybrid crosslinked biomaterials may be a promising candidate for next-generation BHVs.

This study aimed to establish a predictive model to identify children with hematologic malignancy at high risk for delayed clearance of high-dose methotrexate (HD-MTX) based on machine learning. A total of 205 patients were recruited. Five variables (hematocrit, risk classification, dose, SLC19A1 rs2838958, sex) and three variables (SLC19A1 rs2838958, sex, dose) were statistically significant in univariable analysis and, separately, multivariate logistic regression. The data was randomly split into a "training cohort" and a "validation cohort". A nomogram for prediction of delayed HD-MTX clearance was constructed using the three variables in the training dataset and validated in the validation dataset. Five machine learning algorithms (cart classification and regression trees, naïve Bayes, support vector machine, random forest, C5.0 decision tree) combined with different resampling methods were used for model building with five or three variables. When developed machine learning models were evaluated in the validation dataset, the C5.0 decision tree combined with the synthetic minority oversampling technique (SMOTE) using five variables had the highest area under the receiver operating characteristic curve (AUC 0.807 [95% CI 0.724-0.889]), a better performance than the nomogram (AUC 0.69 [95% CI 0.594-0.787]). The results support potential clinical application of machine learning for patient risk classification.

Introduction Drug-resistant cytomegalovirus (CMV) infection remains a challenge in the management of pediatric recipients of hematopoietic stem cell transplantation (HSCT). In this study, we retrospectively reviewed the clinical data on pediatric recipients of HSCT and identified known and unknown drug-resistant CMV variants. Methods A total of 221 children underwent allogeneic HSCT between October 2017 and November 2019 at Shenzhen Children’s Hospital; of these, 35 patients were suspected of having drug-resistant CMV infections and were tested for drug-resistant mutations in the UL97 and UL54 genes by Sanger sequencing. Results Mutations in UL97 or UL54, or in both, were detected in 11 patients. Most of these mutations have not been previously reported. The UL97 mutation (A582V) was detected in only one patient who also harbored two UL54 mutations (T760X and R876W). One patient with both the G604S and T691A mutations in the UL54 gene died of CMV pneumonia. We investigated the risk factors associated with the development of drug-resistant CMV infection. Patients in whom both the donor and recipient had positive CMV serostatuses were less likely to have drug-resistant mutations (Fisher’s exact test, p

Background Gastrointestinal cytomegalovirus (CMV) disease occurs commonly in immunocompromised/immunodeficient patients with advanced human immunodeficiency virus infection, neoplasm, solid organ transplantation, hematopoietic stem cell transplantation, or treatment with immunosuppressants, but is rarely reported in association with measles infection. Case summary We describe a case of extensive gastrointestinal CMV disease secondary to measles infection in a 9-mo-old boy who presented with persistent fever and bloody diarrhea. His condition was improved after ganciclovir treatment. Serological analysis of CMV showed negative immunoglobulin (Ig) M and positive IgG. Blood CMV-DNA was 9.26 × 103 copies/mL. The diagnosis of gastrointestinal CMV disease was confirmed by histopathological findings of intranuclear and intracytoplasmic inclusions and Owl's eye inclusion. This case highlights the differential diagnosis and histopathological characteristics of gastrointestinal CMV infection and laboratory tests. Conclusion Extensive gastrointestinal CMV lesions can be induced by the immune suppression secondary to measles infection. Rational, fast, and effective laboratory examinations are essential for suspected patients.

Asia, the largest continent, is home to 60% of the world’s total population and is the birthplace of many early human civilizations. Throughout history, sport has played an important role in the de...

The enzyme multiplied immunoassay technique (EMIT) is a new method for determining the plasma concentration of voriconazole (VRZ). This study aimed to investigate the correlation between EMIT and high-performance liquid chromatography/ultraviolet rays (HPLC/UV) in determining the plasma VRZ trough concentration in children, in China. A total of 419 blood samples were collected, and plasma VRZ concentrations were detected by the EMIT and HPLC methods. The results of 304 samples were analysed after excluding samples that were undetectable or beyond the quantification limit. A test result value of 0 was defined as undetectable, while concentrations outside the detection range (0.2 - 20.0 μg/ml for HPLC and 0.5 - 16.0 µg/ml for EMIT) were defined as beyond the quantification limit. Results from both methods were compared using the Passing Bablok regression, Bland-Altman plot analysis, and paired Wilcoxon test. The plasma VRZ concentrations determined by EMIT and HPLC showed a strong linear correlation through the linear regression equation

Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children’s Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies.

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naïve and stimulated human CD34+ cells from cord blood (CB) and (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSC population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSC expansion during ex vivo culture.

Background: Next-generation sequencing (NGS) is an emerging technology that can comprehensively assess the diversity of the immune system. We explored the feasibility of NGS in detecting minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) based on immunoglobulin and T cell receptor. Methods: Bone marrow samples were collected pre- and post-treatment with pediatric ALL admitted to Shenzhen Children’s Hospital from February 1st, 2020 to January 31st, 2021. We analyzed the MRD detected by NGS, multiparametric flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR), and analyzed risk factors of positive NGS-MRD at the end of B-ALL induction chemotherapy. Results: A total of paired 236 bone marrow samples were collected from 64 children with ALL (58 B-ALL and 6 T-ALL). The decrease in the clonal rearrangement frequency of IGH, IGK, and IGL was generally consistent after treatment. Positive MRD was detected in 57.5% (77/134) of B-ALL and 80% (12/15) of T-ALL by NGS after chemotherapy, which was higher than those detected by MFC and RQ-PCR. In B-ALL patients, MRD results detected by NGS were consistent with MFC（r = 0.708, p < 0.001）and RQ-PCR（r = 0.618, p < 0.001）. At the end of induction, NGS-MRD of 40.4% B-ALL was >0.01% and multivariate analysis indicated that ≧2 clonal rearrangement sequences before treatment were an independent factor of negative NGS-MRD. Conclusions: NGS is more sensitive than MFC and RQ-PCR for MRD measurement. B-ALL children with ≧2 clonal rearrangements detected by NGS before treatment are difficult to switch to negative MRD after chemotherapy.

Background: Recently, the five-year survival rate of pediatric acute lymphoblastic leukemia (ALL) has reached 90%. However, recurrence remains common following treatment. Recently, chimeric antigen receptor (CAR)-T cell immunotherapy has been shown to be effective in control of relapsed/refractory B-cell malignancies. Although lymphocyte-depleting conditioning chemotherapy, including fludarabine and cyclophosphamide, has increased the efficacy of CAR T-cell therapy, reduction in the tumor load before CAR-T cell infusion is still a challenge. Case presentation: A 12-year-old girl diagnosed with intermediate risk B-ALL in 2017 received chemotherapy according to CCLG-2008 ALL protocol and achieved a complete remission (CR) after induction. However, four years later, she had a first bone marrow relapse and received treatment with HKPHOSG Relapsed ALL 2007 protocol. During maintenance chemotherapy, approximately one year after the first relapse, she developed a second bone marrow relapse with NT5C2 gene mutation detected. Flow cytometry analysis showed that the blasts were CD19 positive. Tumor burden was not well controlled after DEAV (dexamethasone, cytarabine, etoposide) chemotherapy, with the blasts in bone marrow increasing from 49.3% to 96%. The analysis of high-throughput drug sensitivity of tumor resistant genes was consistent with the poor response to chemotherapy. As the leukemia cells progressed rapidly with extremely high burden, CD19 CAR-T cell immunotherapy bridged to hematopoietic stem cell transplantation (HSCT) was introduced at this stage. Following peripheral lymphocyte apheresis, the patient received lymphodepleting conditioning with fludarabine and cyclophosphamide three days before CAR-T cell infusion. Dexamethasone and carfilzomib were then given according to the outcome of high-throughput drug sensitivity test. Around 68.6% blasts were detected by flow cytometry at the day of CAR-T cell infusion. After infusion of CAR-T cells，the patient experienced grade 1 cytokine release syndrome without immune effector cell-associated neurotoxicity syndrome. CR of morphology and molecule biology was achieved on day 28 after CAR-T cell infusion. Finally, the child received haploid-HSCT and remained in remission. Conclusions：Overall, this report reveals that the combination of drug sensitivity test with lymphodepleting conditioning could significantly reduce the tumor burden before infusion of CAR-T cells, and as a result, patients may achieve deeper remission and obtain opportunity for transplantation.Trial registration: clinicaltrials.gov, NCT04016129. Registered 11 July 2019, https://www.clinicaltrials.gov/ct2/show/NCT04016129

Bloodstream infection (BSI) is a serious complication in pediatric hematology-oncology patients. To evaluate the clinical significance of C-reactive protein (CRP), procalcitonin (PCT), albumin, fibrinogen, and D-dimer as potential biomarkers to differentiate among various subtypes of BSIs in pediatric patients with hematological and oncological diseases, we retrieved and analyzed the medical records of pediatric hematology-oncology patients diagnosed with BSI at our hospital between January 2016 and December 2017. The demographic (sex and age) and clinical (primary diseases) characteristics, and laboratory test results (white blood cell and absolute neutrophil counts, and serum CRP, PCT, albumin, fibrinogen, and D-dimer levels) were compared between nosocomial and non-nosocomial; neutropenic and non-neutropenic; and gram-positive and gram-negative BSI episodes. A total of 125 BSI episodes were included, including 69 (55.2%) nosocomial cases, 94 (75.2%) neutropenic cases, and 49 (39.2%) gram-positive episodes. Of the 5 potential biomarkers evaluated (CRP, PCT, albumin, fibrinogen, and D-dimer), PCT levels were significantly lower in neutropenic episodes and gram-positive BSIs (P=0.008 and P=0.001, respectively). At a cutoff value of 0.67 ng/mL, the diagnostic sensitivity, specificity, and positive/negative predictive values of PCT for the differentiation of gram-positive and gram-negative bacterial sepsis were 74.2%, 64.6%, 70.8%, and 65.2%, respectively. We concluded that PCT might potentially serve as a biomarker to differentiate between gram-positive and gram-negative BSIs in pediatric hematology-oncology patients.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is currently the only curative treatment for thalassaemia major (TM). Cord blood (CB) from a sibling has different characteristics from marrow and has potential advantages and disadvantages as a stem cell source.We retrospectively analyzed 68 children with beta-thalassaemia major (β-TM) who underwent fresh cord blood transplantation (F-CBT) from an HLA-matched sibling donor (MSD) between June 2010 and July 2018 in the Department of Pediatrics, Nanfang Hospital and Haematology-Oncology, Shenzhen Children's Hospital.The median infused doses of total nucleated cells (TNCs) and CD34 + cells were 8.51×10The above results indicate that patients with β-TM have excellent outcomes after F-CBT from an HLA-MSD.

Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. However, the underlying mechanisms of the stemness differences because of the cell sources and the culture stimulation are not fully understood. Here, we applied single-cell RNA-seq (scRNA-seq) to analyze the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilized peripheral blood (mPB). We collected over 16,000 single-cell data to construct a comprehensive trajectory inference map and characterized the HSCs population on the hierarchy top, which is under quiescent state. Then we compared HSCs in CB to those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with culture time (CT-SRGs) and cell source (CS-SRGs), respectively. Interestingly, CT-SRGs and CS-SRGs share genes enriched in the signaling pathways such as mRNA catabolic process, Translational initiation, Ribonucleoprotein complex biogenesis and Cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related purine and ATP metabolic process which is important to initiate hematopoiesis. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs and found a small molecule cucurbitacin I, targeting STAT3/JAK2, can efficiently expand HSCs ex vivo while maintaining its stemness. These results indicate SRGs revealed by scRNA-seq can provide helpful insights to understand the stemness differences under diverse circumstances, and CT-SRGs can be a valuable database to identify candidates enhancing functional HSCs expansion during ex vivo culture.

Circular RNAs (circRNAs) are a type of non‑coding RNA with important roles in the regulation of various biological processes involved in malignant progression. However, the potential molecular mechanisms and roles of circRNAs in kidney cancer have remained to be fully elucidated. In a previous study by our group, high‑throughput microarray sequencing data were analyzed to determine the differentially expressed circRNAs in kidney cancer. In this analysis, a novel circRNA (hsa_circ_0100312, named circKL) was identified as a frequently downregulated circRNA in kidney cancer cells and tissues by reverse transcription‑quantitative PCR. In the present study, Cell Counting Kit‑8, colony formation, Transwell, wound‑healing and mouse xenograft assays as well as a lung metastasis experiment were performed to confirm the functions of circKL. The experiments confirmed that circKL overexpression significantly inhibited the proliferation, migration, tumor growth and metastasis of kidney cancer both

Historically, sports were designed by men and for men with the exclusion of women.1 To this day, masculinity is still highly associated with sports. Nevertheless, in Western society, with the advan...

To investigate the genetic etiology, clinical diagnosis and treatment of a child with pancytopenia, failure to thrive and pulmonary infection.Peripheral blood samples of the child and her parents were collected. Genomic DNA was extracted. Genetic variants associated with hematological diseases were detected by high-throughput sequencing.Three variants of TCN2 gene were found, one of which located in exon 5 upstream(c.581-8AT), the parents has carried this variant; one in exon 6 (c.924_927del), the variant was originated from the mother; one in exon 7 (c.973CT), the variant has ocurred de novo. The variants pathogenic analysis combined with clinical manifestation, pancytopenia, the increase in methylmalonic acid level and increased homocysteine, the child was diagnosed with transcobalaminIIdeficiency. The patient presented with respiratory infection, which was confirmed to be pneumocystosis by lung radioscopy and pathogenic high-throughput sequencing of broncho-alveolar lavage fluid. The patient presented with acute respiratory distress syndrome during the treatment with intramuscular injection of vitamin BWe reported a case of Chinese child with TCNII deficiency due to novel gene variant, and analyzed the pathogenicity of the three variants. The treatment of TCNII deficiency with cobalamin should be individualized.

Background Despite much improvement in the treatment for acute lymphoblastic leukemia (ALL), childhood ALLs with MLL-rearrangement (MLL-r) still have inferior dismal prognosis. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. Methods GSE13159 and GSE28497 were selected via the Oncomine website. Differentially expressed genes (DEGs) between MLL-r ALLs and normal samples were identified by R software. Next, functional enrichment analysis of these DEGs were carried out by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Then, the key hub genes and modules were identified by Weighted Gene Co-expression Network Analysis (WGCNA). Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ALL (Phase I) of UCSC Xena analysis, qPCR, and Kaplan-Meier analysis were conducted for validating the expression of key hub genes from bone marrow cells of childhood ALL patients or ALL cell lines. Results A total of 1,045 DEGs were identified from GSE13159 and GSE28497. Through GO, KEGG, GSEA, and STRING analysis, we demonstrated that MLL-r ALLs were upregulating “nucleosome assembly” and “B cell receptor signal pathway” genes or proteins. WGCNA analysis found 18 gene modules using hierarchical clustering between MLL-r ALLs and normal. The Venn diagram was used to filter the 98 hub genes found in the key module with the 1,045 DEGs. We identified 18 hub genes from this process, 9 of which were found to be correlated with MLL-r status, using the UCSC Xena analysis. By using qPCR, we validated these 9 hub key genes to be upregulated in the MLL-r ALLs (RS4;11 and SEM) compared to the non-MLL-r ALL (RCH-ACV) cell lines. Three of these genes, BCL11A, GLT8D1 and NCBP2, were shown to be increased in MLL-r ALL patient bone marrows compared to the non-MLL-r ALL patient. Finally, Kaplan–Meier analysis indicated that childhood ALL patients with high BCL11A expression had significantly poor overall survival. Conclusion These findings suggest that upregulated BCL11A gene expression in childhood ALLs may lead to MLL-r ALL development and BCL11A represents a new potential therapeutic target for childhood MLL-r ALL.

Bloodstream infections (BSI) represent one of the most serious complications in patients in the hematology-oncology unit. In this study, the prevalence, distribution, drug sensitivity profiles, and clinical outcome of BSI were analyzed in pediatric patients with hematological malignancies. Patients admitted to the pediatric hematology-oncology unit at Shenzhen Children's Hospital (Shenzen, China) between January 2016 and December 2017 were enrolled. Their medical records, including gender, age, primary diseases, and microbiology results of all clinical specimens, were reviewed. The incidence of BSI, microbiology characteristics, and effectiveness of antimicrobial therapy were analyzed. A total of 125 BSI cases in 108 patients (mean age, 5.5 years) were recorded, of which 69 (55.2%) were nosocomial BSI cases. The overall rate of BSI was 18.8% in the hematology-oncology unit, of which 75 (75.2%) episodes were neutropenic patients. Patients with nosocomial BSIs and the neutropenic group were older (p#.02, p#.03). HSCTs and AML were more often observed in nosocomial BSIs, while solid tumors were more found in nonnosocomial and non-neutropenic BSIs. BSIs were dominated by Gram-negative pathogens (49.6%) in the hematology-oncology unit compared with Gram-positive pathogens (39.2%). The most common pathogens were coagulase-negative Staphylococcus (24.2%) followed by Klebsiella pneumonia (15.2%), Escherichia coli (12.5%), viridans streptococci (8.2%), and Candida species (7.8%). The antibiotic therapy success rate in patients was 93.5%. Based on our center's experience, Gram-negative pathogens were commonly observed among pediatric hematology-oncology patients with BSI. Coagulase-negative Staphylococcus and K. pneumoniae predominated and antibiotic therapy was effective in these patients.

Our previous study revealed altered resting-stated brain function in children with acute lymphoblastic leukemia (ALL) on new-onset stage. To investigate the effects after induction chemotherapy, a pilot self-contrast study was conducted to compare the difference in resting-stated brain function between pre- and post-induction chemotherapy of ALL. Fractional amplitude of low-frequency fluctuation (fALFF) was employed for fMRI data analysis. Clinical and resting state functional magnetic resonance imaging (RS-fMRI) data of 14 new-onset pediatric ALL patients were collected before and after 3 months of induction chemotherapy. Fourteen age- and gender-matched healthy controls (HCs) were recruited for comparison. Before induction chemotherapy, fALFF values of ALL patients decreased globally, especially in the default mode network (DMN), left frontal lobe, left occipital lobe, and bilateral postcentral gyri as compared to HCs. After induction chemotherapy, fALFF values of ALL patients decreased significantly in the bilateral cuneus, left lingual and calcarine gyri, and left mid frontal gyrus. Paired-sample t-tests and self-contrast analysis showed fALFF increased in the left precuneus, bilateral cuneus, left occipital lobe, bilateral frontal gyri, and bilateral temporal lobes, whereas fALFF in the bilateral precuneus decreased in the ALL patients after induction, which suggests potential side-effects of the treatment. The alteration of fALFF values suggested that resting brain function was impaired before induction chemotherapy and mostly recovered after treatment. This study suggested that fALFF is a reliable and feasible tool in detecting spontaneous brain activity to monitor early neurocognitive impairments in pediatric ALL to better understand the underlying neurobiological mechanisms of chemotherapy on the brain.

Introduction Previous studies have shown that miR-373 functions as either a tumor suppressor or an oncogene depending on which type of cancer it's operating in. However, the functional role of miR-373 in neuroblastoma (NB) remains largely unclear. Methods Expression of miR-373 and SRC kinase signaling inhibitor 1 (SRCIN1) in 20 metastatic and 20 primary NB tissues was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. MTT assay, flow cytometry analysis and transwell migration and invasion assays were performed to evaluate the influence of miR-373 inhibition on the growth, migration and invasion of NB cells, respectively. In vivo experiment was applied to determine the effect of miR-373 inhibition on tumor growth. Dual-luciferase reporter assay was used to confirm the interaction between miR-373 and SRCIN1. Results We observed a significant increase in the expression of miR-373 in metastatic NB samples compared with primary NB samples, and this was inversely correlated with SRCIN1 expression. Functional studies revealed that depletion of miR-373 inhibited in vitro NB cell growth, migration and invasion, and also suppressed tumor growth in an in vivo mouse model. Moreover, we identified that SRCIN1 was a direct and functional target gene of miR-373. Silencing of SRCIN1 partially rescued the antimiR-373-mediated inhibition of cell growth, migration and invasion. Conclusion The data from our study verified a potential oncogenic role of miR-373 in NB cells that occurs through direct targeting SRCIN1. The newly identified miR-373/SRCIN1 axis represents a new potential candidate for therapeutic intervention of malignant NB.

As a research hotspot, circular RNAs (circRNAs) is one type of non-coding RNAs which have many different functions in biological processes. However, there is lack of study investigating the underlying molecular mechanism and the potential roles of circRNAs in Wilms tumor. We conducted a high-throughput microarray sequencing to screen differentially expressed circRNAs in Wilms tumor. A novel circRNA (circ0093740) was identified as a frequently upregulated circRNA in Wilms tumor cells and tissues. Suppression of circ0093740 remarkably inhibited the proliferation and migration ability in Wilms tumor, validated by several experiments. The molecular mechanism of circ0093740 was investigated by luciferase assays and RNA immunoprecipitation assays. The results revealed that circ0093740 promotes the growth and migration ability by sponging miR-136/145 and upregulating DNMT3A. In conclusion, our study discovered the biological role of the circ0093740-miR-136/145-DNMT3A axis in Wilms tumor growth and metastasis which is important for developing new treatment strategy.

Methotrexate (MTX), an antimetabolite for the treatment of leukemia, could cause neutropenia and subsequently fever, which might lead to treatment delay and affect prognosis. Here, we aimed to predict neutropenia and fever related to high-dose MTX using artificial intelligence. This study included 139 pediatric patients newly diagnosed with standard- or intermediate risk B-cell acute lymphoblastic leukemia. Fifty-seven SNPs of 16 genes were genotyped. Univariate and multivariate analysis were used to select SNPs and clinical covariates for model developing. Five machine learning algorithms combined with four resampling techniques were used to build optimal predictive model. The combination of random forest with adaptive synthetic appeared to be the best model for neutropenia (sensitivity = 0.935, specificity = 0.920, AUC = 0.927) and performed best for fever (sensitivity = 0.818, specificity = 0.924, AUC = 0.870). By machine learning, we have developed and validated comprehensive models to predict the risk of neutropenia and fever. Such models may be helpful for medical oncologists in quick decision-making.

Effective systemic treatments for relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) are limited. Recent clinical application of chimeric antigen receptor (CAR) immunotherapy has demonstrated successful control of B-cell malignancies by CAR-T cells; however, designing CARs for T-ALL remains a challenge. CD7 overexpression in T-cell malignancies may be an attractive target for immunotherapy in T-ALL. This study aimed to describe the safe and effective use of autologous CD7-CAR T cells (4SCAR7) for the treatment of T-ALL with induction failure in an 11-year-old patient. Based on The Chinese Children’s Cancer Group-ALL (CCCG-ALL) study protocol, minimal residual disease (MRD) by flow cytometry (FC) analysis was detected on days 19 and 46 of remission induction. At the end of remission-induction chemotherapy, the patient achieved morphologic complete remission, though with MRD 16.13% and RT-PCR of KMT2A-MLLT1 fusion positive, which indicated induction failure. The cerebrospinal fluid (CSF) was negative for blasts at diagnosed. CAR-T therapy and allogeneic transplant were recommended as the next treatment options. CD3+ lymphocytes were collected from the patient 18 days after the high-dose MTX chemotherapy through leukapheresis. The 4SCAR7 CD7-targeting CAR-T cells were generated thereafter. The patient received lymphodepleting chemotherapy prior to 4SCAR7 infusion. Oral administration of itraconazole and sulfamethoxazole was performed from day 0 after CAR-T cell infusion. The patient did not have hypotension, hypoxia, or serious biochemical change or abnormality, but had fever on day 9. Although grade 1 cytokine-release syndrome (CRS) was diagnosed, it was successfully treated with ibuprofen. Anti-CD7 CAR transgene copy numbers in peripheral blood were determined by qPCR, which showed effective expansion initially, then dropped quickly, and persisted at a low level. Although experienced cytopenia from days 14 to 21, the patient achieved remission on day 17. After complete remission, the patient received hematopoietic stem cell transplantation (HSCT) and has recovered well to thisdate. Overall, this report suggested that 4SCAR7 could be a safe and effective strategy for the treatment of pediatric patients with high-risk T-cell malignancies.

β-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for β–thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-ependent β-thalassemia (TDT). However, successful gene therapy for β-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem cells (HSCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q β-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Qrestored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including β0/β0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and the associated risk of tumorigenesis was not observed in cLentiHBBT87Q transduced HSCs through comprehensive analysis. Taken together, we have engineered the cLentiHBBT87Q that can restore β-globin expression in the HSCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.

Background: With the outbreak of coronavirus disease 2019 (2019-nCoV, also named COVID-19) and growing knowledge of its epidemiological characteristics, it has been clear that children with acute lymphoblastic leukemia (ALL) are also susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: In the past four months, we educated the guardians of children with leukemia with a series of preventative measures, including ⑴ Hand Hygiene and Hand Care; ⑵ the use of masks; ⑶ separation and sterilization of patient items; ⑷ avoiding public transportation; ⑸ Staying at home unless medically necessary; ⑹ Telemedicine; ⑺ Allowing only one caretaker during hospital admission ⑻ minimizing patients and their families visiting or travelling. We conducted a retrospective review to study the relationship between compliance with personal preventive measures and exposure to 2019-nCoV among children will ALL and their caretakers before and during the outbreak of 2019-nCoV in China between Jan 21-May 23 of 2020. Results: Prior to the outbreak of coronavirus 2019-nCoV in Jan 2020, 88.9% of patients washed their hands before meals and 55.6% after they ate. All of the patients who are >3 years old and 91% of the patients 3 years old and 78% of the patients Conclusion: From Jan 22 of 2020 to the authorship of this report, within the 200 number of ALL patients whom we have provided care for in Shenzhen Children’s hospital, none of the patients as well as their families were infected with 2019-nCoV, and all of them received chemotherapy as planned. The infection rate of ALL patients suffered febrile neutropenia decreased by seventy-five percent. These outcomes demonstrated that our response to the public health emergency was on-time and appropriate. The operational result shows that the control measures taken are effective. Our experience suggest that offering prompt and appropriate instructions in addition to provision of regular education and preventative practices to patients and their families may be considered to other pediatric oncology centers around the world.