[The Application of RNA-Sequencing in Pediatric B-Cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the molecular genetic characteristics of children with B-cell acute lymphoblastic leukemia (B-ALL) and the application value of RNA-sequencing (RNA-seq). METHODS: The clinical and laboratory examination data of newly diagnosed B-ALL children who were given treatment in the Department of Hematology, Children's Hospital of Chongqing Medical University from May 2015 to April 2020 were collected and analyzed. All children were confirmed by bone marrow morphology, histochemical staining and flow cytometry, and the karyotype analysis, FISH, RT-PCR and RNA-seq detection were conducted. RESULTS: There were 71 males and 58 females with a median age of 50(8-190) months in 129 newly diagnosed children with B-ALL. The fusion gene was positive in 99 children (76.7%). A total of 86 leukemia related or possibly related gene mutations were detected, with a positive rate of 66.7%. There was no significant difference in the detection rates of ETV6-RUNX1, BCR-ABL1, TCF3-PBX1 and KMT2A rearrangements among FISH, RT-PCR and RNA-seq. Rare fusion genes were detected by RNA-seq, including 1 case of KMT2A-USP2, 4 cases of Ph-like related fusion genes, 5 cases of MEF2D rearrangement, 5 cases of PAX5 rearrangement, 3 cases of ZNF384 rearrangement, as well as several fusion genes whose significance were not clear or had not been reported in children with leukemia. Besides, children with ETV6-RUNX1 fusion gene had good response to induction of remission, while children with BCR-ABL1 and ZNF384 rearrangement had poor response, the remission rate of minimal residual disease was statistically significant compared with other types (P<0.05). CONCLUSION: RNA-seq can not only detect known fusion genes, but also discover new or rare fusion genes and gene mutations. The application of RNA-seq has important guiding significance for risk classification and precise targeted therapy of pediatric B-ALL.

Relationship Between Linezolid Exposure and the Typical Clinical Laboratory Safety and Bacterial Clearance in Chinese Pediatric Patients.

Objectives: There have been limited studies concerning the safety and efficacy of linezolid (LZD) in children. This study aimed to evaluate the association between LZD exposure and clinical safety and efficacy in Chinese pediatric patients. Methods: This retrospective cross-sectional study included patients ≤18 years of age who received ≥3 days of LZD treatment between 31 January 2015, and 31 December 2020. Demographic characteristics, medication information, laboratory test information, and bacterial culture results were collected from the Hospital Information System (HIS). Exposure was defined as AUC24 and calculated by the non-linear mixed-effects modeling program (NONMEM), version 7.2, based on two validated population pharmacokinetic models. Binary logistic regression analyses were performed to analyze the associations between AUC24 and laboratory adverse events, and receiver operating characteristic curves were used to calculate the cut-off values. Efficacy was evaluated by bacterial clearance. Results: A total of 413 paediatric patients were included, with an LZD median (interquartile range) dose, duration, clearance and AUC24 of 30.0 (28.1-31.6) mg/kg/day, 8 (4‒15) days,1.31 (1.29-1.32) L/h and 81.1 (60.6-108.7) mg/L·h, respectively. Adverse events associated with TBil, AST, ALT, PLT, hemoglobin, WBC, and neutrophil count increased during and after LZD treatment when compared with before medication (p < 0.05), and the most common adverse events were thrombocytopaenia (71/399, 17.8%) and low hemoglobin (61/401, 15.2%) during the LZD treatment. Patients with AUC24 higher than 120.69 mg/L h might be associated with low hemoglobin 1-7 days after the end of the LZD treatment, and those with an AUC24 higher than 92.88 mg/L∙h might be associated with thrombocytopaenia 8-15 days after the end of the LZD treatment. A total of 136 patients underwent bacterial culture both before and after LZD treatment, and the infection was cleared in 92.6% (126/136) of the patients, of whom 69.8% (88/126) had AUC24/MIC values greater than 80. Conclusion: Hematological indicators should be carefully monitored during LZD treatment, especially thrombocytopaenia and low hemoglobin, and a continuous period of monitoring after LZD withdrawal is also necessary. Since the AUC24 cut-off values for laboratory adverse events were relatively low, a trade-off is necessary between the level of drug exposure required for treatment and safety, and the exposure target (AUC24/MIC) in pediatric patients should be further studied, especially for patients with complications and concomitant medications.

[Serious adverse events associated with chemotherapy in children with acute lymphoblastic leukemia].

OBJECTIVE: To study the occurrence of serious adverse events (SAEs) related to chemotherapy with CCCG-ALL-2015 regimen in children with acute lymphoblastic leukemia (ALL) and the risk factors for death after the SAEs. METHODS: A retrospective analysis was performed on the medical data of 734 children with ALL. They were treated with CCCG-ALL-2015 regimen from January 2015 to June 2019. The occurrence of SAEs during the treatment was investigated. The children with SAEs were divided into a death group with 25 children and a survival group with 31 children. A multivariate logistic regression analysis was used to analyze the risk factors for death after the SAEs. RESULTS: Among the 734 children with ALL, 56 (7.6%) experienced SAEs (66 cases) after chemotherapy, among which 41 cases occurred in the stage of remission induction therapy. Of all 66 cases of SAEs, 46 (70%) were infection-related SAEs, including 25 cases of septic shock (38%), 20 cases of severe pneumonia (30%), and 1 case of severe chickenpox (2%), and 87% of the children with infection-related SAEs had neutrophil deficiency. The most common infection sites were blood and the lungs. The most common pathogens were Gram-negative bacteria, viruses, fungi, and Gram-positive bacteria. There were 16 cases (24%) of hemorrhage-related SAEs, with 11 cases of gastrointestinal bleeding (17%), 4 cases of pulmonary bleeding (6%), and 1 case of intracranial bleeding (2%). Of all 734 children with ALL, 66 (9.0%) died, among whom 25 died due to SAEs. The treatment-related mortality rate was 3.4%, and infection (72%) and bleeding (24%) were the main causes of death. Severe pneumonia was an independent risk factor for treatment-related death in ALL children (OR=4.087, 95%CI: 1.161-14.384, P=0.028). CONCLUSIONS: SAEs often occur in the stage of remission induction therapy, and infection-related SAEs are more common in ALL children accepting chemotherapy with CCCG-ALL-2015 regimen. The development of severe pneumonia suggests an increased risk for death in these children.

Adverse drug events in Chinese pediatric inpatients and associated risk factors: a retrospective review using the Global Trigger Tool.

Understanding the epidemiology and risk factors of adverse drug events (ADEs) in pediatric inpatient is essential if we are to prevent, reduce or ameliorate the harm experienced. The Global Trigger Tool (GTT) is a method of retrospective medical record review that measures harm in hospitalized children. We employed a three-stage retrospective chart review of random samples of 1800 pediatric inpatients discharged from January 2013 to December 2015. 31 kinds of pediatric-specific triggers were made based on the previous trigger tool studies developed for use in adult or pediatric. Positive predictive value (PPV) of individual triggers, as well as ADEs detection rates were calculated. Stepwise logistic regression was performed to investigate risk factors associated with ADEs. Of 1746 patients, detected in 221 patients (12.7%) with 247 ADEs. The PPV of the trigger tool was 13.3%. Of the 247 ADEs, 82.6% were identified as category E, 11.7% category F and 5.7% category H. The pediatric-focused trigger tool is a feasible and useful tool for detecting pediatric ADEs. Especially for patients who have had more drugs, more doses or more admissions which needs to be closely monitored as triggers to improve the safety.

[PRPS1 Expression in Children with Acute Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the correlation between the expression level of PRPS1 and the clinical characteristics in children with acute leukemia(AL). METHODS: Real-time quantitative RT-PCR and Western blot were used to detect the level of PRPS1 mRNA and protein expression in bone marrow samples from 176 patients diagnosed as AL (126 cases were newly diagnosed and 50 cases in complete remission), and its relevance with clinical indicators was statistically analyzed. The bone marrow samples from 21 children with non-malignant hematological disease were used as controls. RESULTS: (1)In B-ALL group, the level of PRPS1 mRNA in newly diagnosed patients were significantly higher than that in control and than that in complete remission patients (both P<0.0001). In T-ALL and AML group, differences was only observed between newly diagnosed patients and complete remission patients(both P<0.0001); (2)In B-ALL group, the expression level of PRPS1 increase with along risk enhancement (P<0.01), while no significant difference was observed in T-ALL (P>0.05). In AML patients, expression difference was shown between low risk group and high risk group(P<0.05); (3)High PRPS1 mRNA expression level were associated with high WBC counts and MRD positive in B-ALL patients (P=0.020, P=0.026, respectively); (4)Expression of NT5C2, an essential gene for relapse and drug resistance, was found to be positively correlated with PRPS1 expression in AL samples(P<0.05). CONCLUSION: High expression of PRPS1 is relevant factor of unfavourable prognosis in B-ALL children, which suggest PRPS1 may be a new indicator for prognosis of pediatric B-ALL and an index to guide individualized chemotherapy.

[Acute leukemia associated with Down syndrome: clinical analysis of 21 cases].

OBJECTIVE: To summarize the clinical characteristics, laboratory findings and prognosis of patients with Down syndrome-related acute leukemia (DS-AL). METHODS: The clinical data, laboratory findings, chemotherapy and prognosis of 21 children with DS-AL were analyzed. RESULTS: Most of the children had disease onset of leukemia at 1 to 5 years of age (85.7%), and acute myeloid leukemia accounted for 57.1% of these cases; 61.9% of the patients had increased lactate dehydrogenase level by 2 folds or more. Of the 13 cases undergoing echocardiaography, 10 (67.9%) showed abnormal findings, and complex congenital heart disease was common (38.5%). Six of the children received chemotherapy and complete remission was achieved in 4 cases; 2 patients died of infection, and the treatment-related mortality was 33.3%. The 2 patients receiving reduced intensive chemotherapy have so far had event-free survival for 21 and 43 months. CONCLUSION: Acute myeloid leukemia is the most common subtype of DS-AL. Patients with DS-AL are sensitive to chemotherapy and the prognosis was favorable with reduced intensive chemotherapy.

Prevalence and genetic analysis of α-thalassemia and ß-thalassemia in Chongqing area of China.

BACKGROUND AND AIMS: Thalassemia is one of the most common hereditary disorders. This study aimed to investigate the prevalence of thalassemia and the mutation spectrum in Chongqing, the southern area of China. METHODS: A total of 1057 children were recruited from Chongqing. Hematological parameters were examined and globin genes were genetically analyzed. RESULTS: The total frequency of thalassemia carriers was 7.76% in this group of children. Among these, α-thalassemia was 5.20%, ß-thalassemia was 1.99% and abnormal hemoglobin variant was 0.57%. Furthermore, 24 cases of α-triplication were detected, frequency of which was 2.55%. The true prevalence of silent α-thalassemia was first reported in this study. In addition, six novel mutations that give rise to α-thalassemia and two rare abnormal hemoglobin variants were first identified in Chinese population. CONCLUSIONS: Our data suggested that the population in Chongqing are at high risk of α- and ß-thalassemia. The findings will be useful for genetic counseling and the prevention of severe thalassemias in this area.

[Respiratory syncytial virus infection in hematopoietic stem cell transplantation recipients with primary immunodeficiencies].

OBJECTIVE: To understand the clinical characteristics and outcome associated with respiratory syncytial virus (RSV) infection in hematopoietic stem cell transplantation (HSCT) recipients with primary immunodeficiencies (PIDs). METHOD: Nasopharyngeal aspirate samples were collected consecutively before and after HSCT from 9 recipients from Apr. 2009 to Sep. 2010 and analyzed for the presence of RSV using real-time polymerase chain reaction assay. To further verify the presence of the virus, positive samples for PCR were isolated for RSV. RSV G gene was amplified, sequenced and used for phylogenetic analysis. RESULT: The presence of RSV was detected in 3 out of 9 children. The viral replication in all the patients was prolonged for months. All the 3 patients with RSV infection were treated with intravenous immune globulin (IVIG) and one was treated with antiviral medication. All patients survived and achieved successful immune reconstitution. CONCLUSION: This study indicates that the HSCT recipients with PID are at increased risk for RSV infection. RSV can shed for months after the initial infection and the patients recover with the course of immune reconstitution.

[Clinical features and outcome analysis of 83 childhood Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with HLH-2004 protocol].

OBJECTIVE: To investigate the clinical features of Epstein-Barr virus-related hemophagocytic lymphohistiocytosis (EBV-HLH), to analysis the outcome of HLH-2004 protocol, and to explore the prognostic factors in EBV-HLH patients. METHODS: The clinical features at onset and outcome of HLH-2004 protocol from 83 pediatric patients with EBV-HLH enrolled from January 2006 to December 2009 in our hospital were analyzed retrospectively. Univariate and multivariate COX regression analysis were used to identify statistically significant prognostic factors. RESULTS: (1) Among the 83 patients, 45 were males and 38 were females. The age of onset ranged from 6 months to 14 years 4 months. 44 patients were treated with HLH-2004, and 3-year overall survival (OS) was (55.8 ± 7.9)%. (2) The most common clinical features of EBV-HLH included high fever, cytopenia, hepatosplenomegaly, and coagulopathy; The respiratory symptoms, angina phlogistic, skin rashes, neurologic abnormality were rare. 97.3% of patients showed an elevation of serum ferritin, liver dysfunction and lipid metabolism disorders was found in most of EBV-HLH patients. 89.0% of patient had hemophagocytosis in bone marrow at diagnosis of EBV-HLH. (3) COX regression analysis revealed that anemia degree, serum albumin < 30 g/L, CD4:CD8 abnormity, NK cell < 3%, treatment protocol were related with the prognosis significantly (P < 0.05). CONCLUSION: EBV-HLH in pediatric patients has severe clinical feature and poor prognosis. HLH-2004 protocol is an effective treatment for patients with EBV-HLH. Symptomatic treatment can't rescue the patients of EBV-HLH.

[Analysis of thirteen cases with secondary coagulation disorder caused by raticide exposure].

OBJECTIVE: To summarize the clinical characteristics of secondary coagulation disorders caused by exposure to poison (raticide) in children and to investigate the diagnosis and corresponding treatment. METHOD: The process of diagnosis, clinical characteristics, response to treatment and the prognosis were analyzed. RESULTS: The main clinical manifestation was mucosal bleeding (66.6%), including epistaxis, gingival bleeding, hematomas and so on. All these children were previously well and had no history of bleeding. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged, factor II was undetectable and the levels of factors VII, IX, and X were lower. The fibrinogen was normal. A raticide was detected in blood and urine of 13 children although 12 of the patients had no definite history of raticide ingestion. Prothrombin complex, fresh frozen plasma and vitamin K(1) were effective in these cases. However, 2 - 3 weeks later, 6 patients presented with recurrent bleeding. CONCLUSION: For children with secondary coagulation disorders of unknown cause, intoxication of raticide should be considered. The administration of blood coagulation factors and vitamin K(1) are effective in early treatment, and the treatment period should be more than 2 months. The PT and APTT should be followed up. Vitamin K(1) should be stopped when PT and APTT are normal.

[Successful treatment of a patient with Wiskott-Aldrich syndrome using hematopoietic stem cell transplantation--case report and literature review].

OBJECTIVE: Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency diseases. The patients with classical WAS have poor prognosis. The hematopoietic stem cell transplantation is the most effective method to cure WAS at present. In this report, a patient with WAS was cured with HLA identical sibling bone marrow transplantation (BMT). METHODS: Wiskott-Aldrich syndrome protein (WASP) was detected using flow cytometry and WASP were analyzed for the diagnosis. The bone marrow was collected from the elder sister who was the HLA identical sibling donor. A total of 4.38x10(8)/kg mononuclear cell (MNC) and 3.78x10(6)/kg CD34+ cells were collected and transfused into the patient after the conditioning regimen with busulfan/cyclophosphamide. Cyclosporine only was used for graft-versus-host disease prophylaxis. WASP and short tandem repeats (STR) were detected as the evidence of engraftment. RESULTS: The diagnosis was WAS: WASP (-IVS9+2T>C, WASP-negative). The patient received busulfan/cyclophosphamide 9 days before the transplantation. WBC decreased to 0.1x10(9)/L in d+4; The absolute number of neutrophils (ANC) was 0.8x10(9)/L in d+13, and exceeded 1.0x10(9)/L later on. From d(-9)-d+14 the patient was dependent on platelet transfusion. From d+15 the patient's PLT>50x10(9)/L and returned to normal after d+30. In d+9-d+10 mild GVHD (I degree) occurred but subsided after the steroid treatment. From d+50, WASP was detected positive and STR showed full donor DNA chimera. Follow-up for 510 d post-transplant, the patient suffered only from mild cold twice, no eczema, no bleeding occurred. The PLT is normal and no chronic GVHD occurred. The levels of IgG, IgM and IgA of the patient were approximately normal. CONCLUSION: The HLA-identical sibling's BMT seems to be the periorit treatment of choice for the WAS patient.

[Hematopoiesis support of mesenchymal stem cells in children with aplastic anemia].

OBJECTIVE: The abnormality of hemopoietic inductive microenvironment (HIM) is involved in the pathophysiology of aplastic anemia (AA). Mesenchymal stem cells (MSC) are main source of bone marrow stromal cells which constitute the bone marrow HIM. Thus, the bone marrow failure in AA may be related to the function of MSC. The aim of the study was to investigate the hematopoiesis support function of MSC in children with AA in vitro. METHODS: Bone marrow samples were collected from 24 children with AA at diagnosis and 19 children with idiopathic thrombocytopenic purpura (ITP), infectious mononucleosis or lymphadenitis (controls). MSCs from bone marrow samples were isolated, cultured and expanded. Morphology, proliferation activity and colony forming unit-fibroblast (CFU-F) were measured. The ability of bone marrow MSC to adhere hemopoietic cells was assayed by MTT. The concentration of stem cell factor (SCF) released from MSC was tested using ELISA. Mononuclear cells (MNC) of bone marrow were plated onto a feeder layer formed by MSC. Cells count and BFU-E, CFU-GM, CFU-GMME productions were measured. RESULTS: The first and third passage time of MSC in children with AA was longer than that in the controls. The number of CFU-F in children with AA (15.70+/-5.78) was less than that in the controls (21.73+/-5.74) (P<0.05). The concentration of SCF in MSC supernatants in children with AA (30.69+/-16.82 pg/mL) was significantly lower than the controls (50.74+/-14.83 pg/mL) (P<0.01). The total MNC count and the number of BFU-E, CFU-GM and CFU-GMME colonies in the support of MSC in children with AA were significantly lower than those in the controls (P<0.01). CONCLUSIONS: The hematopoiesis support function of MSC was significantly reduced in children with AA in vitro. The decreased hematopoiesis support function of MSC may be related its decreased proliferation capacity and SCF release activity.