Unrelated umbilical cord blood transplantation with low-dose anti-thymocyte globulin for children with severe aplastic anemia: A case series.

OBJECTIVE: This study aimed to investigate the prognosis of unrelated umbilical cord blood transplantation (UCBT) using low-dose anti-thymocyte globulin (ATG) in children diagnosed with severe aplastic anemia (SAA). METHODS: This retrospective case series study was conducted involving pediatric SAA patients treated at the Capital Institute of Pediatrics from January 2020 to February 2023. All patients underwent a reduced-intensity conditioning (RIC) regimen alongside low-dose ATG. RESULTS: The study comprised nine patients (five males) with a median age of 5 years (range: 1.7 to 7 years). The median follow-up duration was 799 days (range: 367 to 1481 days), during which all patients survived. The median time interval from diagnosis to transplantation was 3 months (range: 1 to 9 months). The median dosage of ATG administered was 5 mg/kg (range: 2.5 to 7.5 mg/kg). The median durations for granulocyte and platelet engraftment were 15 days (range: 12 to 23 days) and 26 days (range: 12 to 41 days), respectively. Three patients experienced grade 2-4 acute graft-versus-host disease (aGVHD). Epstein-Barr virus (EBV) reactivation was observed in three patients, while cytomegalovirus (CMV) reactivation occurred in seven patients, with no cases of CMV disease or post-transplant lymphoproliferative disorder (PTLD). One patient experienced recurrence 15 months after transplantation due to influenza A infection. CONCLUSION: These findings indicate that SAA patients may attain a favorable prognosis following UCBT with a RIC regimen combined with low-dose ATG.

[Characterization of otorhinolaryngologic manifestations in children with mucopolysaccharide storage disease typeâ and type â ¡].

Objective:To analyze the characteristics of otorhinolaryngological clinical manifestations in children with Mucopolysaccharide（MPS） type Ⅰ and type II in order to improve the knowledge of otorhinolaryngologists about this disease. Methods:Clinical data related to 55 children with MPS type Ⅰ and type II were retrospectively analyzed to investigate the clinical manifestations of MPS in ENT. Results:All 40 patients（72.72%） with MPS had at least one ENT symptom during the course of the disease, with 95% of them having an ENT symptom prior to the diagnosis of MPS; upper airway obstruction was the most common ENT symptom（34, 85.00%）, followed by recurrent upper respiratory tract infections（23, 57.50%）, and lastly, hearing loss（11, 27.50%）; all 26 patients had undergone at least one surgical procedure, of which 15（57.69%） had undergone ENT surgery, and all of these patients underwent ENT surgery before diagnosis. The most common ENT surgery was adenoidectomy. Conclusion:Early clinical manifestations of MPS patients are atypical, but the early and prevalent appearance of otolaryngologic symptoms and increased awareness of the disease among otolaryngologists has a positive impact on the prognosis of MPS.

Somatic ARAF mutations in pediatric Langerhans cell histiocytosis: clinicopathologic, genetic and functional profiling.

ARAF mutations have been identified in a limited subset of patients with Langerhans cell histiocytosis (LCH), a rare disorder characterized by abnormal proliferation of Langerhans cells. LCH is primarily instigated by mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, with BRAFV600E and MAP2K1 mutations constituting most cases. ARAF mutations in LCH highlight the heterogeneity of the disease and provide insights into its underlying molecular mechanisms. However, the occurrence of ARAF-positive LCH cases is extremely rare, with only two reported globally. Although they may be linked to a more aggressive form of LCH and a more severe clinical progression, the clinical significance and functional consequences of these mutations remain uncertain. We performed next-generation sequencing (NGS) to explore driver mutations in 148 pediatric LCH patients and recognized a series of mutations, including an identical novel somatic ARAF mutation, c.1046_1051delAGGCTT (p.Q349_F351delinsL), in four pediatric LCH patients. It was considered an ARAF hotspot mutation. All reported ARAF-positive patients worldwide exhibited characteristic pathological features of LCH, albeit with involvement across multiple systems. In vitro functional studies showed that this mutation could trigger the MAPKinase pathway and phosphorylate its downstream effectors MEK1/2 and ERK1/2 (relatively weaker than BRAFV600E). Over-activation of mutant A-Raf kinase could be inhibited by the BRAF inhibitor vemurafenib. LCH is uncommon, and ARAF mutation is even rarer. In our study, we have identified a novel hotspot somatic ARAF mutation, which has been verified through functional analysis to be an activating mutation. LCH patients with ARAF mutation typically have an unfavorable prognosis due to limited treatment experiences, although they do not exhibit a high relapse rate. To aid in the development of personalized treatment approaches and prognostic markers for LCH patients, it is recommended to conduct typical pathological and immunohistochemical examinations, as well as genetic tests utilizing a targeted gene panel or whole exome sequencing (WES), for LCH diagnosis, thereby promoting the use of inhibitor treatment strategies.

Paediatric autoimmune diseases with ELANE mutations associated with neutropenia.

OBJECTIVE: To explore the clinical characteristics of autoimmune diseases in children with ELANE mutations. METHODS: Three cases of children with ELANE mutations manifesting as autoimmune diseases, who were under treatment from April 2020 to May 2021, were retrospectively analysed. RESULTS: Among the three children, two were boys aged 15 years and 22 months (cases 1 and 3) respectively, and the other one was a 22-month-old girl (case 2). All the cases had recurrent infections. Case 1 presented with cyclic neutropenia and systemic lupus erythematosus (SLE). Case 2 presented with severe neutropenia and autoimmune haemolytic anaemia (AHIA). Case 3 presented with severe neutropenia and anti-neutrophil cytoplasm antibodies (ANCA)-associated small vasculitis. Genetic tests showed that they all had heterozygous mutations in the ELANE gene. Case 1 was treated with methylprednisolone and hydroxychloroquine sulphate for 2 years, making neutrophil level return to normal. Case 2 received allogeneic hematopoietic stem cell transplantation and has stopped taking antibiotics, steroids and all the immunosuppressors. Case 3 received subcutaneous injections of granulocyte colony-stimulating factor, oral prednisone and cyclophosphamide. The boy in case 3 has been followed up for one year, and his absolute neutrophil count has increased to 1.56 × 109/L. CONCLUSION: Patients with ELANE mutations, combined with autoimmune diseases, may have recurrent infections. Disease-modifying antirheumatic drugs (DMARDs) are effective for autoimmune diseases. Autoimmune diseases with ELANE mutations associated with neutropenia can be cured through allogeneic hematopoietic stem cell transplantation.

A genomic DNA-based NGS method for the simultaneous detection of multiple fusion genes in pediatric leukemia.

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA-, instead of RNA-, based next-generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS-270) with an RNA-based multiplex reverse transcription-PCR technique with a detection panel of 57 fusion genes (MRTP-57). MRTP-57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up-front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS-270 and MRTP-57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS-270 over MRTP-57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP-57 were also identified by MTCS-270, clearly signifying the respectable detection accuracy of MTCS-270. Notably, across the patients screened, MTCS-270 identified more samples with fusion genes than MRTP-57, illustrating a broader fusion gene detection coverage by MTCS-270. The present study provides solid evidence that this DNA-based NGS approach can be used as a potential detection tool together with other well-established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS.

Analysis of Multiple Vitamins Serum Levels and Disease-Related Factors in Children with Acute Leukemia.

Objective: To explore the relationship between vitamins levels and disease-related indicators in children with acute leukemia (AL). Methods: A total of 107 hospitalized children with AL were enrolled in this study and assigned to one group in each of the following categories: infected group (n = 52) and noninfected group (n = 55); treatment remission group (n = 56) and nonremission group (n = 51); high-risk (HR) group (n = 44), intermediate risk (IR) group (n = 53), and slight risk (SR) group (n = 8); cyclophosphamide + cytosine arabinoside+6-mercaptopurine + pegaspargase group (CAML, n = 15); methotrexate group (MTX, n = 9); and vindesine + daunomycin + L-asparaginasum + prednisone (VALP, n = 38). Hematological and serological parameters, hepatic and renal function, and changes in vitamins A, B1, B2, B6, B9, B12, C, D, and E serum content in children with AL were analyzed to investigate their relationship with AL disease-related factors. Results: The vitamin D level was significantly higher in the noninfected group than in the infected group (P < 0.05). Compared with the nonremission group, the level of vitamin B1 in the treatment remission group was significantly higher, while the levels of vitamin B6 and B12 were notably lower (P < 0.05). The levels of vitamins B6 and B12 were notably different among the treatment groups. Multivariate analysis showed that hemoglobin (Hb) and C-reactive protein (CRP) were predisposing factors of AL in children. The disease type (acute lymphoblastic leukemia/acute myelogenous leukemia) was the factor affecting remission in AL children. Abnormal kidney function and the occurrence of icterus were the influencing factors for the risk degree in AL children. Platelet (PLT) count, activated partial thromboplastin time (APTT), neutrophils (N), and immunophenotype were shown to affect the choice of therapeutic regimens. Conclusion: There are notable vitamins imbalances in children with AL. The imbalances influence disease-related factors and therefore provide some references for the prognosis and treatment of AL.

Clinical outcomes of individualized busulfan-dosing in hematopoietic stem cell transplantation in Chinese children undergoing with therapeutic drug monitoring.

To identify relationships between busulfan (Bu) exposure and outcomes of a cohort pediatric patients receiving hematopoietic stem cell transplantation (HSCT), along with a targeted busulfan-based conditioning regimen. We retrospectively evaluated targeted busulfan concentrations in 53 pediatric patients (age 0.4-16 years) who received busulfan 4 times daily according to recommended weight-based doses in a single-center analysis between 2018 and 2020. In this trial, individual busulfan pharmacokinetics were performed following dose 5 of the conditioning regimen. Twenty four of 53 patients (45.3%) studies did not require dose adjustments. Equal number of patients (24/53) required one dose adjustments while two-dose adjustment applied for 5 of 53 (9.4%). Twenty-one percent of the patients exhibited ll-lV aGVHD. The incidence of veno-occlusive disease (VOD) was in 3.8% of the 53 patients, while incidence of hemorrhagic cystitis (II-III) reached to 9.7%. Engraftment was successful in 98% of the 53 patients with relapse in 2% of cases. The probability of overall survival and disease-free survival at day 100 was 96% and 94%, respectively. In conclusion, therapeutic drug monitoring (TDM) and individualization of Bu dosage are essential to improve the efficacy and safety of busulfan-based regimen in Chinese pediatric HSCT recipients.

Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. RESULTS: We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. CONCLUSIONS: This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

Next-Generation Sequencing Revealed Disease-Causing Variants in Two Genes in a Patient With Combined Features of Spherocytosis and Antley-Bixler Syndrome With Genital Anomalies and Disordered Steroidogenesis.

Conventionally, patients with combined rare diseases are often difficult to diagnose. This is because some clinicians tend to consider the multiple disease symptoms as the presentation of a complicated "syndrome." This pattern of thinking also confines their way of filtering pathogenic mutations. Some real pathogenic mutations might be ignored due to not covering all disease presentations. Here we report the case of a girl who was suffering from spherocytosis and Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis. She remained undiagnosed even after targeted gene detection before. However, after performing next-generation sequencing and analyzing the sequencing data, we identified two mutations: c.2978T > A in ANK1 and c.1370G > A in POR. Our findings and experiences in diagnosing these mutations could contribute to the existing knowledge on the clinical and genetic diagnosis of patients with disease presentations in multiple systems.

Posttransplant cyclophosphamide for haploidentical stem cell transplantation in children with Wiskott-Aldrich syndrome.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is the curative treatment for Wiskott-Aldrich syndrome (WAS). However, it is difficult to find a matched donor for patients. Therefore, haploidentical donors should be considered for patients lacking a suitable donor. Our pilot study evaluated whether HSCT with posttransplantation cyclophosphamide (PTCy) is an effective treatment for WAS. METHODS: Haploidentical family donors were selected as donor sources for a total of five patients without a suitable donor between March 2015 and March 2017. A modified transplant protocol using PTCy (50 mg/kg/day on days +3 and +4) was performed, including busulfan (16 mg/kg), fludarabine (150 mg/m2 ), and rabbit antihuman thymocyte globulin (7.5 mg/kg). RESULTS: The median time for neutrophil recovery over 1,000 × 103 /mm3 was 15 days (range, 12-18 days), and that for keeping platelets counts over 50,000/mm3 was 27.5 days (range, 20-35 days). The median follow-up was 2.1 years (range, 1.4-2.5 years). Two patients developed grade I acute graft-versus-host disease (GVHD), and one patient had limited chronic GVHD. All five patients are alive and independent of platelet infusion with 100% donor chimerism. CONCLUSION: Our pilot study suggests that HSCT with modified PTCy is a safe and effective treatment for WAS, which needs further clinical practice and research.

Posttransplant Cyclophosphamide for HLA-haploidentical Transplantation in Patients With Mucopolysaccharidosis.

We successfully used a haploidentical transplantation protocol with posttransplant cyclophosphamide (CY) (50 mg/kg/d on days +3 and +4) for in vivo T-cell depletion in patients with mucopolysaccharidosis using reduced-intensive conditioning regimens, followed by a busulfan-based conditioning regimen, which included busulfan (12 to 16 mg/kg) and fludarabine(150 to 200 mg/m)+rabbit antihuman thymocyte globulin (7.5 to 10 mg/kg) as a conditioning regimen. Cyclosporine or tacrolimus, methotrexate, mycophenolate mofetil, and methylprednisolone were administered to prevent graft-versus-host disease (GVHD). After follow-up for a median period of 1.5 years, all 8 patients without preexisting severe comorbidities and early transplant referrals are alive, with 100% donor chimerism and excellent performance status. Only 1 patient developed chronic GVHD(II). We conclude that posttransplant CY is effective in vivo for T-cell depletion to promote full donor engraftment in patients with mucopolysaccharidosis. In addition, with posttransplant CY, the procedure reduced the rate of GVHD and the cost of transplant and improved the patients' quality of life.

Evaluation of antitumor activity of survivin short interfering RNA delivered by lipid nanoparticles in colon cancer in vitro and in vivo.

Survivin has been overexpressed in numerous types of cancer and is associated with a poor clinical outcome. A number of various approaches have been used to counteract survivin in order to inhibit tumor growth or promote cell apoptosis. The present study aimed to evaluate the efficiency and antitumor effect of a survivin-targeted short interfering RNA (siRNA) delivery system using lipid nanoparticles for the treatment of colon cancer. Survivin siRNA (si-survivin) nanoliposomes were prepared and transfected into LoVo cells. The mRNA expression level of survivin was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Cell viability was evaluated by MTT assay. LoVo-bearing nude mice were treated with si-survivin intratumorally or intravenously. Tumor growth in LoVo-bearing mice was monitored and recorded, and tumor samples were obtained for evaluation of survivin expression levels using RT-qPCR, western blotting and immunohischemical staining. The expression level of survivin was significantly reduced by nanoliposomal si-survivin along with cell proliferation inhibition in vitro. Intravenous administration of si-survivin nanoliposomes may significantly inhibit tumor growth with less toxicity compared with doxorubicin hydrochloride treatment in LoVo-bearing mice. Nanoliposomal si-survivin may significantly reduce the expression level of survivin and inhibit cell proliferation of colon cancer cells in vitro and in vivo. si-survivin delivered by lipid nanoparticles may be a potential treatment approach for colon cancer.

Small interfering RNA targeting of the survivin gene inhibits human tumor cell growth in vitro.

The present study aimed to evaluate the impact of small interfering RNA (siRNA) targeting of the survivin gene in human tumor cells and the effect of decreased survivin expression on the proliferation and apoptosis of tumor cells. Human tumor cell lines (MSA-MB-231, SGC-7901, HeLa, A549, SK-OV-3 and Raji, PC-3) were cultured in vitro and divided into three groups: survivin siRNA-treated, scrambled negative control siRNA-treated and an untreated control group. The level of survivin mRNA and protein expression was subsequently determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation was also examined by an MTT assay following transfection and the apoptotic rate of cells was detected by Hoechst and Annexin V/propidium iodide staining. It was observed that relative to the control group, expression of survivin mRNA and protein in the survivin siRNA-treated group was significantly downregulated. Furthermore, siRNA targeting of survivin lead to the inhibition of tumor cell proliferation, as well as an increase in their apoptotic rate in vitro. These data suggest that survivin may be a potential tumor biomarker and a novel target for the treatment of cancer.

Survivin downregulation using siRNA nanoliposomes inhibits cell proliferation and promotes the apoptosis of MHCC-97H hepatic cancer cells: An in vitro and in vivo study.

At present, survivin is one of the most cancer-specific proteins that has been identified. The present study aimed to investigate the antitumor effects of novel survivin small interfering RNA (siRNA) nanoliposomes targeting survivin in human hepatocellular carcinoma MHCC-97H cells and xenograft mouse models. Survivin-targeted siRNA nanoliposomes were prepared and transfected into MHCC-97H cells and MHCC-97H-bearing nude mice. Survivin expression was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Cell viability was analyzed using an MTT assay and apoptosis was evaluated using Hoechst and Annexin V-fluorescein isothiocyanate/propidium iodide staining. Tumor growth in MHCC-97H-bearing mice was monitored following treatment and tumor samples were obtained for survivin expression analysis using RT-qPCR, western blotting and immunohistochemistry staining. Survivin expression levels were significantly downregulated by nanoliposome-mediated survivin siRNA delivery and this was associated with a significant inhibition of cell growth and an increase in the apoptosis of MHCC-97H cells. Downregulation of survivin expression using survivin siRNA nanoliposomes inhibited tumor growth in the MHCC-97H xenograft models without significant treatment-associated toxicity. Therefore, a cationic nanoliposome-based survivin siRNA delivery system was constructed and demonstrated to be efficient for survivin siRNA delivery in in vitro and in vivo studies. These results demonstrate that survivin downregulation was able to significantly attenuate cell proliferation and induce the apoptosis of MHCC-97H cells, as well as inhibit tumor cell growth in MHCC-97H xenograft models, indicating that survivin suppression using siRNA may contribute to the inhibition of tumor development by suppressing cell proliferation and promoting apoptosis.

A correlation analysis of miRNA­34a and its predicted target genes in leukemia.

microRNA­34a (miRNA­34a) plays an important role in the pathogenesis of leukemia. This study aimed to explore its role in the proliferation of HL­60 cells and the correlation with some of its predicted target genes: the cyclin­dependent kinase 4 (CDK4), the oncogene MYB and the silent information regulator 1 (SIRT1). We first analyzed the expression of miR­34a, CDK4, MYB and SIRT1 in peripheral blood samples from acute leukemia (AL) patients and healthy controls, and conducted a correlation analysis. HL­60 cells were then transfected with miR­34a and control 'scramble̓ miRNA, and quantitative RT­PCR and western blotting were used to analyze the effects of the interfering sequence in HL­60 cells. The expression of miR­34a was significantly reduced in AL patients compared to healthy controls (P<0.01), and negatively correlated with the expression of CDK4 and MYB. Sub­group analysis revealed that the expression of MYB was significantly lower in AL children <3 years old compared to those >3 years. Following the transfection of HL­60 cells with miR­34a, the mRNA level of CDK4, MYB and SIRT1 decreased by 53.2, 43.3 and 33.5%, respectively, compared to the control, similarly to the respective changes in protein levels. This study showed that the expression of miR­34a negatively correlates with the expression of CDK4 and MYB in pediatric patients with acute leukemia. miRNA­34a downregulates the expression of the CDK4, MYB and SIRT1 genes in vitro; it may thus represent a novel therapeutic target for acute leukemia.

[Suppression of survivin gene in leukemia cells by small interfering RNA].

OBJECTIVE: To evaluate the impact of specific siRNA on survivin gene in transfected leukemia cells. METHOD: The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into K562 cell by Hiperfect into human leukemia cell line K562, which has high survivin expression level. The level of survivin mRNA expression was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR) with SYBR GREEN I. The apoptosis index of cytotrophoblasts were determined and analyzed by FCM (Annexin V-FITC/PI staining methods). The cell proliferation was examined by MTT at 48 h and 72 h after transfection. RESULT: The level of mRNA expression was significantly inhibited by the siRNA 48 h and 72 h after transfection, the suppression rate of survivin mRNA separately reached 85.21%, 94.35% mensurated by quantitative RT-PCR with SYBR GREEN I, cell proliferation was inhibited significantly by 45.02% and 50.88%, respectively, the apoptotic rate detected by Annexin V-FITC assay reached 12.28%and 21.55%, respectively. CONCLUSION: The chemosynthesized siRNA targeting survivin could significantly down-regulate survivin mRNA. Survivin siRNA was able to inhibit the proliferation of leukemia cell line K562. Survivin may become a new target for leukemia gene therapy.