The potential role of RNA sequencing in diagnosing unexplained insensitivity to conventional chemotherapy in pediatric patients with B-cell acute lymphoblastic leukemia.

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is a highly heterogeneous disease. According to large-scale RNA sequencing (RNA-seq) data, B-ALL patients can be divided into more than 10 subgroups. However, many genomic defects associated with resistance mechanisms have not yet been identified. As an individual clinical tool for molecular diagnostic risk classification, RNA-seq and gene expression pattern-based therapy could be potential upcoming strategies. In this study, we retrospectively analyzed the RNA-seq gene expression profiles of 45 children whose molecular diagnostic classifications were inconsistent with the response to chemotherapy. The relationship between the transcriptome and chemotherapy response was analyzed. Fusion gene identification was conducted for the included patients who did not have known high-risk associated fusion genes or gene mutations. The most frequently detected fusion gene pair in the high-risk group was the DHRSX duplication, which is a novel finding. Fusions involving ABL1, LMNB2, NFATC1, PAX5, and TTYH3 at onset were more frequently detected in the high-risk group, while fusions involving LFNG, TTYH3, and NFATC1 were frequently detected in the relapse group. According to the pathways involved, the underlying drug resistance mechanism is related to DNA methylation, autophagy, and protein metabolism. Overall, the implementation of an RNA-seq diagnostic system will identify activated markers associated with chemotherapy response, and guide future treatment adjustments.

[Effect of Low-Dose Recombinant Interleukin-2 Therapy on Immunocyte Subsets in Children with Solid Tumor].

OBJECTIVE: To evaluate the effect of low-dose recombinant interleukin-2 (rIL-2) therapy on immunocyte subsets and its side effects in children with solid tumor. METHODS: A total of 22 children (11 males and 11 females) with solid tumor in our department from December 2012 to November 2017 were selected, with a median age of 9 (3-16) years old when starting IL-2 therapy. ALL surgeries and chemotherapy of children had been completed before low-dose rIL-2 therapy, and 17 cases achieved complete remission (CR) and 5 cases achieved partial remission (PR). A low-dose rIL-2 therapy was given 1 month after chemotherapy for 1 year: 4×105 IU/(m2·d), s.c. for every other day, 3 times per week. The immunocyte subsets were detected every 3 months until the end of treatment, meanwhile, disease condition and therapy-related side effects were followed up. RESULTS: After low-dose rIL-2 therapy in 22 children, the absolute values of CD3+ T cells, CD3-CD56+ natural killer cells, CD3+CD4+ helper T cells (Th) and CD3+CD8+ cytotoxic T cells were up-regulated remarkably, as well as Th/suppressor T cells (all P < 0.05). While, there were no significant differences in absolute value and proportion of CD4+CD25+CD127- Treg cells during therapy. Among the 17 children who achieved CR before rIL-2 therapy, 14 cases continued to maintain CR after therapy, while 3 cases relapsed, and with 2 died after treatment abandonment. The 5 children who achieved PR before low-dose rIL-2 therapy were evaluated CR by PET/CT scan after treatment. In the early stage of low-dose rIL-2 therapy, 1 child developed skin rashes at the injection sites, and 2 children ran a slight to mild transient fever. Their symptoms disappeared without any organ damage after symptomatic treatment. CONCLUSION: Low-dose rIL-2 therapy has good drug tolerance, and changes the distribution of anti-tumor immune-cell subgroup in peripheral blood of children with solid tumor remarkably without up-regulation of absolute value and ratio of Treg cells.

Local Treatment of Children Suffering From Parameningeal Rhabdomyosarcoma: A Retrospective Single-Center Study From China.

BACKGROUND: Treatment for parameningeal rhabdomyosarcoma (PM-RMS) has been a challenge since local control is difficult. The goal of this study was to analyse the impact of different local treatment approaches on childhood PM-RMS patients and help dispel the doubt that whether secondary radical surgery (SRS) should be encouraged in the management of PM-RMS. METHODS: A total of 17 children with PM-RMS who received unified systemic chemotherapy and individualized local therapy such as radiotherapy (RT) and/or SRS were included in this retrospective study. The overall survival (OS) and event free survival (EFS) were compared between groups adopting different local strategies. RESULTS: The 3-year OS and EFS of our PM-RMS patients was 75.5% and 56.5% respectively. The OS and EFS of patients who received SRS were both significantly lower than that of the non-SRS group (3-year OS: 50.0% vs 90.0%, P = .031; 3-year EFS: 33.3% vs 60.6%, P = .020). The OS and EFS of the patients who received RT was higher than that of the patients of the non-RT group (3-year OS: 85.6% vs 0%, P = .001; 3-year EFS: 64.0% vs 0%, P = .011). CONCLUSION: This study illustrates that SRS was associated with poor prognosis of PM-RMS and should not be routinely performed. Optimized RT strategies along with more intensive chemotherapy may be alternative options to improve the survival of patients with PM-RMS. Multi-center, large sample and prospective studies are needed to further validate these findings.

Vemurafenib combined with chemotherapy achieved sustained remission in pediatric LCH: a multi-center observational study.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a myeloid neoplasia with potentially fatal consequences, and about 2/3 of cases involve the BRAFV600E kinase-activated mutation. Vemurafenib, a BRAF inhibitor, has demonstrated significant clinical improvements in LCH. However, the high relapse rate of LCH following cessation of vemurafenib therapy remains a major challenge, and alternative treatment strategies require further investigation. METHODS: In this retrospective multi-center study, we evaluated the efficacy and safety of vemurafenib combined with conventional chemotherapy in patients with severe or refractory LCH. RESULTS: Seventeen patients were enrolled in the study, with eleven classified as risk organ involvement (RO +). Six received the combination therapy as the primary treatment, and eleven after being refractory to prior chemotherapy. The overall response rate was 94.1%. Progression-free survival among all 17 patients was 70.6% (12/17) at a median follow-up of 32 months, and relapse-free survival among the 15 patients with discontinuation after a response was 73.3%(11/15) at a median follow-up of 34 months. Five of six patients (83.3%) with myeloid BRAFV600E mutations demonstrated molecular remission. The overall survival rate was 100%. Adverse events were mostly classified as grades 1 or 2. CONCLUSION: Our data suggest that the combination of vemurafenib and chemotherapy can achieve sustained clinical and molecular level relief in children with LCH, and side effects are tolerable.

Arsenic trioxide induces ferroptosis in neuroblastoma by mediating GPX4 transcriptional inhibition.

Neuroblastoma (NB), the most common extracranial solid tumor in childhood, significantly contributes to cancer-related mortality, presenting a dearth of efficacious treatment strategies. Previously, our studies have substantiated the potent cytotoxicity of arsenic trioxide (ATO) against NB cells, however, the specific underlying mechanism remains elusive. Here, we first identified ATO as a novel GPX4 inhibitor, which could trigger the ferroptosis in NB cells. In vitro, ATO significantly inhibited the proliferation and migration ability of NB cells SK-N-AS and SH-SY5Y, and induced ferroptosis. Furthermore, the iron chelator deferoxamine reversed ATO-mediated intracellular reactive oxygen species accumulation and hindered the generation of the lipid peroxidation product malondialdehyde. Conversely, ferric ammonium citrate notably intensified its cytotoxic effects, especially on retinoic acid (RA)-resistant SK-N-AS cells. Subsequently, the quantitative real-time polymerase chain reaction results showed ATO significantly inhibited the transcription of GPX4 in NB cells. Remarkably, immunoblotting analysis revealed that MG132 exhibited a notable effect on elevating GPX4 levels in NB cells. Nevertheless, pretreatment with MG132 failed to reverse the ATO-mediated decrease in GPX4 levels. These findings suggested that ATO reduced the GPX4 expression level in NB cells by mediating GPX4 transcriptional repression rather than facilitating ubiquitinated degradation. In conclusion, our research has successfully indicated that ATO could induce ferroptosis and initiate lipid peroxidation by regulating the transcriptional repression of GPX4, and ATO holds promise as a potential anti-tumor agent in NB, specifically for patients with RA-resistant HR-NB.

Arsenic Trioxide inhibits Activation of Hedgehog Pathway in Human Neuroblastoma Cell Line SK-N-BE(2) Independent of Itraconazole.

BACKGROUND: Neuroblastoma (NB) remains associated with a low overall survival rate over the long term. Abnormal activation of the Hedgehog (HH) signaling pathway can activate the transcription of various downstream target genes that promote NB. Both arsenic trioxide (ATO) and itraconazole (ITRA) can inhibit tumor growth. OBJECTIVE: To determine whether ATO combined with ITRA can be used to treat NB with HH pathway activation, we examined the effects of ATO and ITRA monotherapy or combined inhibition of the HH pathway in NB. METHODS: Analysis of CCK8 and flow cytometry showed cell inhibition and cell cycle, respectively. Real-time PCR analysis was conducted to assess the mRNA expression of HH pathway. RESULTS: We revealed that as concentrations of ATO and ITRA increased, the killing effects of both agents on SK-N-BE(2) cells became more apparent. During G2/M, the cell cycle was largely arrested by ATO alone and combined with ITRA, and in the G0/G1 phase by ITRA alone. In the HH pathway, ATO inhibited the transcription of the SHH, PTCH1, SMO and GLI2 genes, however, ITRA did not. Instead of showing synergistic effects in a combined mode, ITRA decreased ATO inhibitory effects. CONCLUSION: We showed that ATO is an important inhibitor of HH pathway but ITRA can weaken the inhibitory effect of ATO. This study provides an experimental evidence for the clinical use of ATO and ITRA in the treatment of NB with HH pathway activation in cytology.

Serum brain-derived neurotrophic factor (BDNF) as predictors of childhood neuroblastoma relapse.

BACKGROUND: Neuroblastoma (NB) is a childhood malignant tumor,50% of high-risk NB children still have recurrence, and the long-term survival rate is very low. NB tumors expressing high levels of BDNF/TrkB are associated with poor survival outcomes.In this study, we show that the trends of serum concentration of BDNF at different growth stages after birth, and explore the relationship with NB replase. METHODS: In experiment 1, 87 subjects were enrolled and divided into four groups, neonates group、 children group、adults group and NB patients. The distribution of serum concentration of BDNF by ELISA. In experiment 2, we studied BDNF in stage 4 NB patients to determine their frequency, correlation with clinical parameters, and prognostic impact. RESULTS: First, we identified that serum BDNF concentration decreased from the newborn to childhood in healthy subjects, while it was relatively high in children(age > 1 year) with NB. In the second phase our studies showed no significant increase in serum BDNF concentration in these NB patients, with adverse pathologic features, large tumor maximum diameter, and MYCN amplification. After comprehensive treatment, levels of BDNF gradually increased in children with recurrence and decreased in the remission group. High serum BDNF concentration was associated with relapse. Of 21 stage 4 neuroblastoma patients, adopted a comprehensive treatment approach including ATO-basic modified chemotherapy, traditional radiotherapy,stem cell transplatation and immunotherapy. 76% of alive patients having > 3 years follow-up. CONCLUSION: The aim is to show that BDNF is a predictor of recurrence risk of NB.

Prognostic value and outcome for acute lymphocytic leukemia in children with MLL rearrangement: a case-control study.

PURPOSE: To evaluate the prognostic factors and outcome for acute lymphoblastic leukemia (ALL) in children with MLL rearrangement (MLL-r). METHODS: A total of 124 pediatric patients who were diagnosed with ALL were classified into two groups based on the MLL-r status by using a retrospective case-control study method from June 2008 to June 2020. RESULTS: The prevalence of MLL-r positive in the whole cohort was 4.9%. The complete remission (CR) rate on Day 33 in the MLL-r positive group was not statistically different from the negative group (96.8% vs 97.8%, P = 0.736). Multivariate analysis showed that T-cell, white blood cell counts (WBC) ≥ 50 × 109/L, MLL-AF4, and D15 minimal residual disease (MRD) positive were independent risk factors affecting the prognosis of MLL-r positive children. Stem cell transplantation (SCT) was a favorable independent prognostic factor affecting event-free survival (EFS) in MLL-r positive patients (P = 0.027), and there was a trend toward an independent prognostic effect on overall survival (OS) (P = 0.065). The 10-year predicted EFS for patients with MLL-AF4, MLL-PTD, MLL-ENL, other MLL partner genes, and MLL-r negative cases were 46.67 ± 28.61%, 85.71 ± 22.37%, 75 ± 32.41%, 75 ± 32.41%, and 77.33 ± 10.81%, respectively (P = 0.048). The 10-year predicted OS were 46.67 ± 28.61%, 85.71 ± 22.37%, 75 ± 32.41%, 75 ± 32.41%, and 85.2 ± 9.77%, respectively (P = 0.049). The 124 patients with ALL were followed up and eventually 5 (4%) cases relapsed, with a median relapse time of 3.9 years. CONCLUSION: Patients with MLL-r positive ALL have moderate remission rates, but are prone to relapse with low overall survival. The outcome of MLL-r positive ALL was closely related to the partner genes, and clinical attention should be paid to screening for MLL partner genes and combining them with other prognostic factors for accurate risk stratification.

Arsenic trioxide increases apoptosis of SK-N-BE (2) cells partially by inducing GPX4-mediated ferroptosis.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial tumor in central nervous system threatening children's health with limited therapeutic options. Arsenic trioxide (ATO) has been identified the cytotoxicity in NB cells but the potential mechanism remains unclear. In this study, we attempted to obtain some insight into the mechanisms of cell death induced by ATO in NB cells. METHODS AND RESULTS: Proteomic analyses found that ATO can affect the signaling pathway associated with ferroptosis, including the upregulation of iron absorption (FTL, FTH1, HO-1), ferritinophagy (LC3, P62, ATG7, NCOA4) and modifier of glutathione synthesis (GCLM); downregulation of glutamine synthetase (GS) and GPX4, which was the critical inhibitor of ferroptosis. Western blot analysis revealing GPX4 expression in SK-N-BE (2) cells decreased after treatment with ATO (7.3 µM), resulting in a loss of GPX4 activity. Furthermore, Ferroptosis inhibitor ferrostatin-1 partially blocked ATO-induced cell death. CONCLUSIONS: Our study revealed that ATO may induce ferroptosis in neuroblastoma cell SK-N-BE (2) by facilitating the downregulation of GPX4, ultimately resulting in iron-dependent oxidative death.

Encouraging Early Outcomes of Treatment With Arsenic Trioxide Combined With Chemotherapy for Alveolar Rhabdomyosarcoma in Children: 4 Case Reports.

BACKGROUND: Alveolar rhabdomyosarcoma (ARMS) is a subtype of rhabdomyosarcoma characterized by its aggressive behavior and poor prognosis, highlighting the need for novel treatment options. Arsenic trioxide (ATO) has been shown to specifically inhibit tumor growth and the metastasis of ARMS in vitro by acting on the hedgehog pathway. Here we report on a pilot clinical study to evaluate the activity of an ATO-combined chemotherapy approach for the treatment of ARMS patients. METHODS: We designed a therapeutic schedule of an ATO-combined chemotherapy, incorporating comprehensive management according to the Intergroup Rhabdomyosarcoma Study Group protocol. ATO was administered at 0.16 mg/kg per day over 8 h via an IV for 10 days combined with a chemotherapeutic regimen of vincristine, actinomycin, and cyclophosphamide (VAC regimen) on the third day, which was repeated every 21 days. A total of eight cycles of ATO-combined chemotherapy were applied throughout the entire scheme. RESULTS: A total of three refractory/recurrent and one untreated ARMS patient, three male and one female, with a median age of 5.8 years (range, 5.1 to 12.5 years), were enrolled in the present study. All patients were sensitive to combined chemotherapy with ATO and achieved partial or complete remission during treatment. Except for reversible gastrointestinal reaction and myelosuppression, no other adverse events were observed during the process of treatment. CONCLUSIONS: The combined chemotherapy of ATO and the VAC regimen exhibited beneficial activities against ARMS in pediatrics and was well tolerated, but prospective large-scale clinical trials are warranted to determine the long-term efficacy, optimal courses, and late toxicity in this population.

Treatment barriers and clinical outcome of children with medulloblastoma in China: a report from the Chinese Children's Cancer Group (CCCG).

BACKGROUND: Medulloblastoma (MB) is the most common malignant central nervous system tumor of childhood. Management requires interdisciplinary care and is associated with unique challenges in developing regions. Here, we report the characteristics, clinical outcome and treatment barriers for Chinese children with MB based on a multi-institutional cohort from the Chinese Children's Cancer Group (CCCG). METHODS: Retrospective cohort study among 12 Chinese pediatric oncology units from the CCCG Brain Tumor Workgroup on patients aged <18 years diagnosed with MB from 2016 to 2019. RESULTS: 221 patients (male:female = 138:83) were included, 175 (79%) were ≥3 years of age, and 46 (21%) <3 years. 177 patients (80%) were completely staged, among which 50 (28%) had metastasis and 70 (40%) were considered to have high-risk (HR) disease. Gross/near-total resection was achieved in 203 patients (92%). In patients where molecular grouping could be assigned, 19 (16%), 35 (29%), and 65 (54%), respectively had WNT-activated, SHH-activated, and Group 3/4 MB. The median duration between resection and initiation of adjuvant therapy was 36 days. Respective 2-year PFS and OS rates were 76.0 ± 3.0% and 88.0 ± 2.3%. PFS was significantly associated with age, metastatic status and clinical risk grouping. Chemotherapy use during CSI or alkylator choice were not significant predictors for patient outcome. CONCLUSIONS: We reported the clinical profiles and outcome from the largest cohort of Chinese children with MB after multi-modal therapy. Strengths and limitations on the local provision of neuro-oncology service are identified.

Improved Outcomes with Induction Chemotherapy Combined with Arsenic Trioxide in Stage 4 Neuroblastoma: A Case Series.

Objective: The apoptotic and cytotoxic effects of arsenic trioxide (ATO) makes it a potentially suitable agent for the treatment of patients with neuroblastoma with poor prognosis; therefore, we try to evaluate the effectiveness and safety of ATO combined with reinduction/induction chemotherapy in children with recurrent/refractory or newly diagnosed stage 4 neuroblastoma. Methods: Retrospective analysis was performed on seven pediatric patients with recurrent /refractory or newly diagnosed stage 4 neuroblastoma treated with traditional reinduction/induction chemotherapy combined with ATO. Results: A total of 7 patients were treated synchronously with ATO and chemotherapy for up to nine courses; all patients received conventional chemotherapy plus a 0.16 mg/kg/day dose of intravenous ATO during reinduction/induction chemotherapy. Treatment was effective in five patients and ineffective in the other two patients. The overall response rate was 71.43% (5 of 7). The side effects of the ATO combination were minor, whereby only treatment in one patient was terminated at the sixth course due to a prolonged QT interval (0.51 s), which returned to normal after symptomatic treatment. Conclusions: ATO can be safely and effectively combined with chemotherapy drugs as a potential alternative means of treatment for high-risk stage 4 neuroblastoma, and we have observed that ATO can restore the sensitivity of chemotherapy in some patients who were resistant to previous chemotherapy. Further investigations and clinical data are required to confirm these observations.

Salidroside alleviates taurolithocholic acid 3-sulfate-induced AR42J cell injury.

OBJECTIVES: To investigate the protective effects of Salidroside (Sal) on AP cell model induced by taurolithocholic acid 3-sulfate (TLC-S) as well as its underlying mechanism. METHODS: AR42J cells were divided into normal group (N group), AP cell model group (Mod group), Sal treated alone group (S+N group) and Sal treated AP cell model group (S+Mod group). The cell viability was examined by CCK-8 assay. Secretion of lipase and trypsin by AR42J cells, quantified using commercial assay kits, was used as the markers of TLC-S-induced pancreatitis. The levels of TNF-α, IL-1ß, IL-8, IL-6 and IL-10 in the cell supernatant were measured by ELISA. The effect of Sal on molecules in the NF-κB signaling pathway and autophagy was investigated by qRT-PCR and western blot. RESULTS: The decreased cell viability in Mod group was increased by Sal (P < 0.01). The upheaved activities of lipase and trypsin in AP cell model were declined by Sal (P < 0.01). The levels of TNF-α, IL-1ß, IL-8 and IL-6 in the cell supernatant, Beclin-1 and LC3-Ⅱ mRNA and protein, p-p65/p65 protein, which were increased in AP cell model, were decreased by Sal; and IL-10 in the cell supernatant, LAMP2 mRNA and protein, p-IκBα/IκBα protein which was declined in AP cell model, was increased by Sal (P < 0.05 or 0.01). There were no significant differences in all indexes between the N and S+N groups (P > 0.05). CONCLUSIONS: Sal alleviated AR42J cells injury induced by TLC-S, inhibited the inflammatory responses and modulated the autophagy, mainly through inhibiting the NF-κB signaling pathway.

Immune Microenvironment in Langerhans Cell Histiocytosis: Potential Prognostic Indicators.

In this study, the immune microenvironment in Langerhans cell histiocytosis (LCH) was characterized to determine if immune indices are predictive of severity. Serum samples from 54 treatment-naïve patients were analyzed quantitatively for inflammatory cytokines and immunoglobulins before and after the induction of chemotherapy. The initial serum sIL-2R, TNF-α, and IL-10 of untreated LCH patients with risk organ involvement (RO+) were significantly higher than those with single-system (SS) involvement. LCH patients with hematologic involvement exhibited a significantly higher sIL-2R, TNF-α, IL-10, and IL-1ß expression, as compared to the group without involvement. sIL-2R, TNF-α, and IL-10 were increased in patients with liver or spleen involvement. Th cells have decreased in the liver+ and spleen+ group, and Ts cells were significantly decreased in non-response group after induction chemotherapy. The serum level of immune indices represents, to some extent, the severity of the disease. Pertinent laboratory inspections can be used to improve risk stratification and guide immunotherapy.

Wilms Tumor 1 Mutations Are Independent Poor Prognostic Factors in Pediatric Acute Myeloid Leukemia.

The prognostic impact of Wilms tumor 1 (WT1) mutations remains controversial for patients with acute myeloid leukemia (AML). Here, we aimed to determine the clinical implication of WT1 mutations in a large cohort of pediatric AML. The clinical data of 870 pediatric patients with AML were downloaded from the therapeutically applicable research to generate effective treatment (TARGET) dataset. We analyzed the prevalence, clinical profile, and prognosis of AML patients with WT1 mutations in this cohort. Our results showed that 6.7% of total patients harbored WT1 mutations. These WT1 mutations were closely associated with normal cytogenetics (P<0.001), FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD) mutations (P<0.001), and low complete remission induction rates (P<0.01). Compared to the patients without WT1 mutations, patients with WT1 mutations had a worse 5-year event-free survival (21.7 ± 5.5% vs 48.9 ± 1.8%, P<0.001) and a worse overall survival (41.4 ± 6.6% vs 64.3 ± 1.7%, P<0.001). Moreover, patients with both WT1 and FLT3/ITD mutations had a dismal prognosis. Compared to chemotherapy alone, hematopoietic stem cell transplantation tended to improve the prognoses of WT1-mutated patients. Multivariate analysis demonstrated that WT1 mutations conferred an independent adverse impact on event-free survival (hazard ratio 1.910, P = 0.001) and overall survival (hazard ratio 1.709, P = 0.020). In conclusion, our findings have demonstrated that WT1 mutations are independent poor prognostic factors in pediatric AML.

Eltrombopag as first-line treatment for thrombocytopenia among paediatric patients after allogeneic haematopoietic stem cell transplantation.

AIMS: The purpose of this study is to examine the safety and efficacy of eltrombopag as first-line treatment for thrombocytopenia among paediatric patients after haematopoietic stem cell transplantation (HSCT). METHODS: Forty-three childhood patients with thrombocytopenia after HSCT who received eltrombopag were retrospectively analysed. RESULT: Eltrombopag was began at the median of 27 days after HSCT and lasted for 24 days. Thirty-five children responded to eltrombopag therapy, and the cumulative platelet recovery rate was 88.9%. The cumulative incidence of platelet recovery was lower (83.9 vs 100%; P = .035) in patients with decreased numbers of megakaryocytes before starting eltrombopag than in those with normal. Factors associated with a significantly elevated response to eltrobopag from univariate analysis were donor type. Results from the multiple regression analysis found that weight (hazard ratio [HR] = 0.7, 95% confidence interval [CI] 0.5-0.9, P = .022), platelet engraftment time (HR = 1.0, 95%CI 1.0-1.0, P = .012) and bone marrow megakaryocytes (HR = 8.0, 95%CI 1.5-43.3, P = .016) before starting eltrombopag were the independent risk factors. Based on Youden's index algorithm in the receiver-operating characteristic curve, the optimal cut-off value of the maintenance dose of eltrombopag in predicting nonresponders was 4 mg/kg. The area under the receiver-operating characteristic curve was 0.923 with sensitivity of 97.8%, specificity of 87.9%, positive predictive value of 72.3%, and negative predictive value of 92%. None of the paediatric patients stopped using eltrombopag due to side effect or intolerability. CONCLUSION: Eltrombopag is effective and safe in paediatric patients with thrombocytopenia after HSCT. The number of megakaryocytes in bone marrow before eltrombopag treatment may serve as a predictor of the response to eltrombopag. We recommend that the maintenance dose of eltrombopag should not exceed 4 mg/kg/d.

Prediction, management, and prognosis of mixed chimerism after hematopoietic stem cell transplantation in transfusion-dependent pediatric thalassemia patients.

BACKGROUND: Early-onset mixed chimerism (MC) with a high proportion of residual host cells is considered a signal of graft rejection in patients undergoing allogenic hematopoietic stem cell transplantation for transfusion-dependent thalassemia. In order to prevent graft rejection and minimize the risk of treatment-related graft-versus-host disease (GVHD), we established a hierarchical management system based on chimerism analysis. METHOD: This retrospective study provides a comprehensive review of the characteristics, interventions, and outcomes of the 38 patients who developed MC after transplantation among the 144 pediatric thalassemia patients between July 2007 and January 2019 at our center. RESULTS: A sibling donor, a blood type-matched donor, conditioning regimens without fludarabine, and transplants containing <10 × 108 total nucleated cells/kg were identified to be associated with the development of MC. Among the 38 patients developing MC, only four patients rejected the grafts. The response rate to donor lymphocyte infusion (DLI, only for patients receiving sibling donor transplantation) and cytokine immunomodulation without DLI was 70.6% and 42.9%, respectively. Patients that developed GVHD after DLI or cytokine therapy had a more significant increase in donor cell chimerism (16%, range 0%-35%) than those without (8.5%, range -21% to 40%, P = .049). However, even when treatment-related GVHD was included, patients with MC had a lower cumulative incidence of total acute GVHD than patients with complete donor chimerism (29.2% vs 48.0%, P = .030). CONCLUSIONS: Interventions based on chimerism analysis were effective in preventing graft rejection and did not increase treatment-related GVHD in thalassemia patients with MC.

[Efficacy of BMMSCs on aGVHD and Its Correlation with SerumInflammatory Cytokines in Pediatric Patients with Severe Refractory Acute Graft-Versus-Host Disease].

OBJECTIVE: To investigate the efficacy of bone marrow mesenchymal stem cells (BMMSC) on children with refractory graft-versus-host disease (GVHD) and to judge the efficacy of BMMSC by dynamically monitoring the changes of cytokines in children with GVHD before and after infusion of BMMSC, so as to provide a theoretical basis for clarifying the mechanism of BMMSC. METHODS: 17 children with refractory aGVHD including 7 of grade II, 6 cases of grade III and 4 cases of grade IV after allo-HSCT were enrolled. All the children with aGVHD, who received routine immunosuppressive therapy, but the state of disease not improved, were treated with immunosuppressive drugs combined with BMMSC infusion. Study endpoints included safety of BMMSC infusion, response to BMMSC, and overall response of aGVHD. The serum levels of IL-2α, IL-6, IL-10, IL-8 and TNF-α in aGVHD patients were measured by chemiluminescence before infusion of BMMSCs and Day 7, Day 14 after infusion of BMMSCs. RESULTS: The cumulative median dose of BMMSCs was 5.5 (3.4-11.1) × 106/kg for average of 3.7 times, and the median time of 16.5 (4-95) days for the first infusion of MSCs. In 17 cases of refractory GVHD, 14 responded to treatment, whereas 3 patients failed. The total effective rate was 82.4% and no adverse reactions occurred. Of the 14 survived cases (82.4%), the median follow-up time was 944 (559-1245) days from the first infusion of MSCs. The levels of TNF-α in children with grade II, III and IV GVHD before treatment were 9.5±4.3 pg/ml, 16.3±10.9 pg/ml and 35.8±21.2 pg/ml respectively. The difference between grade II and IV, III and IV was statistically significant (P<0.05). Compared with the ineffective group of BMMSC infusion, the serum TNF-αlevel in the BMMSCs treatment effective group was 10.8±5.6 pg/ml vs 40.6±14.8 pg/ml (t=-3.901, P<0.05) before treatment. In the effective group of BMMSCs infusion, IL-10 20±17.4 pg/ml of day 14 was significantly higher than that 7.3±3.1 pg/ml before the treatment (t=-2.850, P<0.05), while , the serum levels of IL-2α, IL-6, IL-8, TNF-α were not statistically significantly different (P＞0.05). CONCLUSION: The infusion of BMMSC is safe and effective in the treatment of refractory GVHD in children. TNF-αlevel relates with the severity of GVHD. BMMSC may play an anti-GVHD role by up regulating the level of cytokine IL-10 in vivo.

Cytokine-induced killer cells/natural killer cells combined with anti-GD2 monoclonal antibody increase cell death rate in neuroblastoma SK-N-SH cells.

Neuroblastoma (NB) is one of the most common extracranial, solid, pediatric malignancies. Despite improvements in conventional therapies, including surgery, chemotherapy and radiation therapy, the prognosis of stage IV NB remains poor, indicating that novel treatment strategies are required. Immunotherapies, such as anti-GD2 monoclonal antibodies, used alone or in combination with cytokines, and peripheral blood mononuclear cells or cord blood mononuclear cells (CBMNCs), have been indicated to cause NB cell death and to prolong patient survival in high-risk NB; however, they remain limited by severe cytotoxicity and side effects. In the present study, it was determined that anti-GD2 monoclonal antibody alone or CBMNC-isolated cytokine-induced killer (CIK)/natural killer (NK) cells alone significantly induced cell death of NB SK-N-SH cells, and the combination of anti-GD2 antibody and CIK/NK cells could significantly increase the cell death rate compared with either treatment alone. In addition, based on a method referred to our previous study, it was identified that a two-cytokine culture system, using interleukin IL-2 and IL-7, effectively stimulated the proliferation of CIK/NK cells. These results serve to suggest a novel treatment strategy for relapsed/refractory NB with high efficiency and few side effects.

Pre-application of arsenic trioxide may potentiate cytotoxic effects of vinorelbine/docetaxel on neuroblastoma SK-N-SH cells.

BACKGROUND: Arsenic trioxide is effective in the treatment of acute promyelocytic leukemia and is currently in use in clinical trials for the treatment of solid tumor types. Given that arsenic trioxide is able to arrest neuroblastoma cell cycle in the G2/M phase, the present study is, to the best of our knowledge, the first to investigate whether the combination of arsenic trioxide with mitosis-phase-specific antineoplastic agents (vinorelbine or docetaxel) or non-mitosis-phase-specific antineoplastic agents (etoposide or cisplatin) exert synergistic effects in cytotoxicity on the human SK-N-SH neuroblastoma cell line. METHODS: Neuroblastoma cells were either incubated with one of the four drugs individually, or preincubated with arsenic trioxide and then followed by another drug when cell cycle was arrested at the G2/M phase with the highest proportion. RESULTS: The results of the present study revealed that arsenic trioxide potentiated the apoptotic rate of neuroblastoma cells induced by chemotherapeutic drugs. The present study further demonstrated that preincubation with arsenic trioxide followed by a mitosis-phase-specific antineoplastic agent result in a higher cytotoxicity effect compared with a non mitosis-phase-specific antineoplastic agent. Along with the enhanced cytotoxicity in combination group, the cell cycle distribution demonstrated a decreased proportion of G2/M phase in the combination group. CONCLUSION: The in vitro study revealed that the pre-application of arsenic trioxide followed by mitosis-phase-specific antineoplastic agents potentiate the cytotoxic effects on neuroblastoma cells, therefore arsenic trioxide may be a promising therapeutic option for treating neuroblastoma.