[The Factors Affecting Relapse in Pediatric B-cell Acute Lymphoblastic Leukemia Patients without Prognostic Fusion Genes Following Up for 10 years].

OBJECTIVE: To analyze the efficacy of children with B-cell acute lymphoblastic leukemia (B-ALL) without prognostic fusion genes treated by CCLG-ALL 2008, and investigate the related factors affecting the recurrence of the patients. METHODS: B-ALL patients without prognostic fusion genes treated by the protocol of CCLG-ALL 2008 in our hospital from March 2008 to December 2012 were retrospectively analyzed. Follow-up time was ended in August 31, 2019. The median follow-up time was 92 months (range 0-136 months). Kaplan-Meier was used to detect the RFS, and COX multivariate regression analysis was employed to identify the independent factors affecting the recurrence of the patients. RESULTS: There were 140 males and 99 females enrolled in this study. The ratio of male to female was 1.41∶1. The median age was 4.4 years old and the median number of WBC at initial stage was 4.98×109/L. There were 77 cases relapsed during the observation while 162 without relapsed, 16 cases lost to follow-up and 72 cases died. The recurrence and mortality rate was 32.22% and 30.1%, respectively, in which 45 cases died of recurrence (62.5% of the total deaths). Univariate analysis showed that the age≥6 years old, WBC >100×109/L, the bone marrow blasts on day 15≥25%, the bone marrow minimal residual disease (MRD) at week 12 >10-4, and the higher risk were the main factors affecting the recurrence of the patients (P<0.05). Multivariate COX regression analysis showed that age≥6 years old, WBC >100×109/L, bone marrow MRD >10-4 at the 12th week were the independent risk factors affecting recurrence of the patients. CONCLUSION: Age, initial WBC, and bone marrow MRD at the 12th week were correlated with recurrence in children with B-ALL without prognostic fusion genes, which can be used as prognostic indices of recurrence risk in clinical.

[Expression and Clinical Significance of Long Non-coding RNA AC002454.1 in Children with Acute Leukemia].

OBJECTIVE: To study the difference of long non coding RNA (lncRNA) expression profile in bone marrow specimens of children with acute leukemia (AL) and other hematological disease children with normal bone marrows as controls, to screen the lncRNA related with childhood hematological diseases, and to explore the expression of lncRNA AC002454.1 and its clinical significance in AL children. METHODS: The microarray gene chip technology was used to statistically analyze the lncRNA in bone marrow cells of newly diagnose AL children and control children. Ninty-seren differentially expressed lncRNAs were selected. The bone marrow specimens of ALL children (21 cases), AML children (22 cases) and control children (21 cases) were verified and compared by using qRT-PCR; then the lncRNA with maximum differential expression-lncRNA AC002454.1 was selected and used to analyze the relation of relative expression level with clinical indicators. RESULTS: The microarray gene chip detection showed that 1 884 differentially expressed lncRNA were found in ALL children, and 4 289 differentically expressed lncRNA were found in AML children. The results confirming these differentically expressed lncRNA by qRT-PCR showed that 9 lncRNA expression were significantly up-regulated in ALL children, and 12 lncRNA expression were significantly up-regulated in AML children. Among these up-regulated lncRNA, the difference of AC002454.1 expression was most significant in ALL and AML children (P＜0.05, P＜0.01). The detection showed that there was a significant difference, in AC002454.1 relative expression level of newly diagnosed T-ALL and B-ALL children (P＜0.01), moreover, this difference also was found in ALL and AML children (P＜0.05). The detection analysis showed that there was no statistical difference in AC002454.1 relative expression level among the different sex, age, WBC count at initial diagnosis, chromosome, fusion gene, and risk stratification (P＞0.05 for all). CONCLUSION: The lncRNA expression profile of AL children has been gained by using the lncRNA microarray gene chip technicology. AC002454.1 the significantly high expression exist in AL children, which relates with immunotyping and prognosis of AL children in a certain degree.

[Safety and Efficacy of VDMP Re-induction Regimen in Chinese Children with Relapsed Acute Lymphoblastic Leukemia-A Report from Jiangsu Childhood Hematology Group].

OBJECTIVE: To evaluate the therapeutic safety and efficacy of VDMP re-induction regimen in Chinese children with relapsed acute lymphoblastic leukemia (ALL). METHODS: Forty-one patients with relapsed ALL were prospectively enrolled in this study. All the patients were distributed in 3 children's hospitals and treated with VDMP regimen as the first re-induction chemotherapy. Therapeutic efficacy and side-effects were analyzed. RESULTS: The ratio of male to female was 27:14. The median age was 7.9 (2.2-15.4) years old. Patients relapsed at very early, early, and late stage were 7 cases, 11 cases, and 23 cases, respectively.The immunophenotype analysis showed that 38 cases were B-ALL, and 3 cases were T-ALL. All patients suffered from grade 4 of neutropenia and forty(97.6%) cases got infection, of them one case died. Thirty-nine(95.1%) cases had nonhematologic adverse event at least one organ involved grade 3 in 38 out of 41 cases, the VDMP therapy was completed, 34(89.5%) cases achieved a complete remission (CR), 1 case achieved partial remission(PR), and 3 cases didn't get remission. Follow-up data of 38 cases with completing VDMP chemotherapy were obtained, only one case was lost. Among 37 cases available for evaluation, 16 cases received allo-hematopoietic stem cell transplantation(allo-HSCT) after chemotherapy, and 13 patients survived, while 21 cases did not receive allo-HSCT(treated with chemotherapy only), and 8 patients survived.The overall survival rate of allo-HSCT group was significantly higher than that of those treated with chemotherapy only(P<0.05). CONCLUSION: VDMP re-induction regimen is effective and well tolerable for pafients in the treated children with relapsed ALL. After remission, allo-HSCT is recommended with the aim of long survival.

Outcome of children with newly diagnosed acute lymphoblastic leukemia treated with CCLG-ALL 2008: The first nation-wide prospective multicenter study in China.

Acute lymphoblastic leukemia (ALL) is the most common malignancy among children. The trial Chinese Children Leukemia Group (CCLG)-ALL 2008 was a prospective clinical trial designed to improve treatment outcome of childhood ALL through the first nation-wide collaborative study in China. Totally 2231 patients were recruited from ten tertiary hospitals in eight cities. The patients were stratified according to clinical-biological characteristics and early treatment response. Standard risk (SR) and intermediate risk (IR) groups were treated with a modified BFM based protocol, and there was 25%-50% dose reduction during intensification phases in the SR group. Patients in high risk (HR) group received a more intensive maintenance treatment. Minimal residual disease (MRD) monitoring with treatment adjustment was performed in two hospitals (the MRD group). Complete remission (CR) was achieved in 2100 patients (94.1%). At five years, the estimate for overall survival (OS) and event-free survival (EFS) of the whole group was 85.3% and 79.9%, respectively. The cumulative incidence of relapse (CIR) was 15.3% at five years. The OS, EFS and CIR for the SR group were 91.5%, 87.9%, and 9.7%, respectively. The outcome of the MRD group is better than the non-MRD group (5y-EFS: 82.4% vs 78.3%, P = .038; 5y-CIR: 10.7% vs 18.0%, P < .001). Our results demonstrated that the large-scale multicenter trial for pediatric ALL was feasible in China. Dose reduction in the SR group could achieve high EFS. MRD-based risk stratification might improve the treatment outcome for childhood ALL.

Gene polymorphisms in the folate metabolic pathway and risk of pediatric acute lymphoblastic leukemia: a case-control study in a Chinese population.

Polymorphisms in folate pathway genes may influence susceptibility to pediatric acute lymphoblastic leukemia (ALL). This case-control study was undertaken to analyze the association of genetic polymorphisms (677C>T and 1298A>C) of methylenetetrahydrofolate reductase (MTHFR) and reduced folate carrier (RFC1) (80G>A) with the risk of pediatric ALL in China. A total of 176 pediatric ALL patients and 170 matched healthy subjects (as controls) were included and DNA was extracted from the peripheral blood. SNaPshot single nucleotide polymorphism typing was used to determine the genotypes of MTHFR 677C>T, MTHFR 1298A>C, and RFC1 80G>A. All statistical analyses were conducted with SAS software (version 9.2; SAS Institute). There were no significant differences in the genotype and allele frequencies of MTHFR 677C>T, MTHFR 1298A>C, or RFC1 80G>A between patients and controls. No significant correlation was found between the combined genotypes of these polymorphisms and the risk of developing ALL in this study. Furthermore, no significant differences were observed for 677C>T and 1298A>C frequencies between the control and case groups. There was no association between MTHFR 677C>T, MTHFR 1298A>C, or RFC1 80G>A gene polymorphisms and risk of pediatric ALL in the Han Chinese population.

[Clinical Features and Prognostic Factors of Children with Acute Lymphoblastic Leukemia in High-Risk Group].

OBJECTIVE: To explore the clinical features and prognostic factors of pediatric acute lymphoblastic leukemia (ALL) in high-risk (HR) group. METHODS: A total of 421 children with ALL in the Children's Hospital of Soochow University from August 2008 to March 2013 were diagnosed and treated according to the Chinese Children Leukemia Group (CCLG)-2008 Protocol. Among different risk-groups, 148 cases were stratified into the low-risk group and 191 cases were included in the moderate-risk group. Eight-two patients of the high-risk group were analyzed retrospectively for their clinical features, 5-year event-free survival (EFS) rate and overall survival (OS) rate. RESULTS: The median follow-up times of 82 patients were 64 months(3.0-76.3 months), 55 patient achieved complete remission(CR) after 1 cycle of induction chemotherapy(CR rate 67.1%), 25 patients relapsed(30.5%) mainly in very early and early relapse phases, significantly different from the low-risk group (P=0.013), 27 pateitns died(32.9%). The 5-year pEFS and pOS were 57.20% and 58.5%, respectively. Ph+ or BCR/ABL+ and MRD>10-2 on the 33rd day in the high-risk group were 2 main factors influencing EFS and OS according to single factor analysis. Ph+ or BCR/ABL+ was an independent prognostic factor, however, the MRD value on the 33rd day was not statistically significant differente by virtue of COX regression analysis. CONCLUSION: The clinical feature of children with ALL in high risk group display low induction CR rate, high recurrence rate and the lower 5-year pEFS. Ph+ or BCR/ABL+ is regarded as an independent factor of poor prognosis.

mRNA expression profiling of histone modifying enzymes in pediatric acute monoblastic leukemia.

Histone modification is dysregulated in various types of cancers, including hematological malignancies. However, the expression profile of histone-modifying enzymes in pediatric acute monoblastic leukemia (AML FAB M5) has not been investigated. In this study, we evaluated the mRNA expression profile of 85 genes that encode enzymes involved in histone-modification in 27 pediatric AML FAB M5 samples by using a novel real-time PCR array. We obtained a gene cluster consisting of a total of 28 genes (15 up-regulated genes and 13 down-regulated genes). This gene signature revealed up-regulated expression of putative oncogenes GCN5L2, SETD8, KDM5C, AURKA and AURKB, and downregulated putative tumor suppressor genes (TSGs) EP300, PRMT3, PRMT8 and NOTCH2. We investigated possible biological interactions between differentially expressed genes using ingenuity pathway analysis (IPA) and found 12 significant networks. Among these, gene expression, cancer, and embryonic development showed the highest number of networks with 39 focus molecules and had an associated significance score of 68. Further, Rb, CDKN2C, and E2F1 were found to be upstream regulators of histone-modifying enzymes. This study provides additional insights into the molecular pathogenesis of pediatric AML FAB M5. These genes represent interesting targets with potential for diagnostic, prognostic and therapeutic application in pediatric AML patients.

E2A-PBX1 exhibited a promising prognosis in pediatric acute lymphoblastic leukemia treated with the CCLG-ALL2008 protocol.

OBJECTIVE: The objective of this study was to observe the prognosis of pediatric patients with E2A-PBX1-positive acute lymphoblastic leukemia (ALL) from the treatment with the CCLG-ALL2008 protocol. DESIGN AND METHODS: Three hundred and forty-nine Chinese pediatric patients with pre-B-cell ALL were enrolled in this study from December 2008 to September 2013. Of these, 20 patients with E2A-PBX1 expression and 223 without the gene expression were stratified into two cohorts. Clinical and biological characteristics and 5-year event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS) were analyzed and compared between these two groups. RESULTS: The E2A-PBX1 fusion transcript was detected in 20 of 349 (5.7%) patients. Compared with the gene-negative subgroup, patients with E2A-PBX1 were younger in age but did not show significant differences in white blood cell (WBC) count or gender distribution at primary diagnosis. Moreover, there were more inferior karyotypes detected in the E2A-PBX1 subgroup (P=0.035). With the CCLG-ALL2008 treatment protocol, patients with E2A-PBX1 showed a favorable treatment response with lower minimal residual disease (MRD) levels (<10-4) at time point 1 (TP1, P=0.039) but no superior steroid response or histological remission. We also observed a promising survival outcome, with a 5-year EFS reaching 95.0%±4.9% versus 66.3%±3.9% in the gene-negative group (P=0.039). However, we did not find significant differences in RFS (P=0.061) and OS (P=0.113). CONCLUSION: Our data provided clinical observation of Chinese pediatric patients. Patients with E2A-PBX1-positive ALL benefited well from the CCLG-ALL2008 protocol, a risk-based intensified treatment trial, with lower levels of MRD and longer RFS duration though they had no favorable characteristics at primary diagnosis.

Clinical significance of microRNA-34b expression in pediatric acute leukemia.

The present study aimed to explore the function of miR­34b promoter methylation in cell proliferation in children's acute leukemia. Quantitative PCR and methylation­specific PCR were performed to measure the levels of miR­34b and its promoter methylation in normal cells, eight leukemia cell lines as well as primary leukemic cells isolated from patients newly diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lymphocytic lymphoma. miR­34b levels in leukemia cell lines and primary leukemic cells were significantly lower than those in normal cells. The miR­34b promoter was found to be methylated in all leukemia cell lines, 24 of 31 ALL patients and 8 of 19 AML patients, but not in the 23 normal controls. miR­34b expression and methylation of its promoter were not associated with most clinical parameters assessed; however, miR­34b levels in prednisone­sensitive ALL were significantly different from those in insensitive ALL. A cell counting kit­8 assay showed that transfection of miR­34b mimics into K562 cells inhibited their proliferation. Furthermore, treatment with the demethylating agent 5­aza­2­deoxycytidine significantly enhanced miR­34b expression levels and decreased the methylation status of its promoter in HL­60 and K562 cells. In conclusion, the results of the present study indicated that in pediatric leukemia cells and leukemia cell lines, the expression of miR­34b is inhibited by methylation of its promoter, which impairs the restraining effects of miR­34b on cell proliferation. It was also indicated that the expression of miR­34b in ALL patients may affect their response to early treatments.

Microarray profiling of bone marrow long non-coding RNA expression in Chinese pediatric acute myeloid leukemia patients.

Long non-coding RNA (lncRNA) plays a role in gene transcription, protein expression and epigenetic regulation; and altered expression results in cancer development. Acute myeloid leukemia (AML) is rare in children; and thus, this study profiled lncRNA expression in bone marrow samples from pediatric AML patients. Arraystar Human LncRNA Array V3.0 was used to profile differentially expressed lncRNAs in three bone marrow samples obtained from each pediatric AML patient and normal controls. Quantitative polymerase chain reaction (qRT-PCR) was performed to confirm dysregulated lncRNA expressions in 22 AML bone marrow samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to construct the lncRNA-mRNA co-expression network. A total of 372 dysregulated lncRNAs (difference ≥10-fold) were found in pediatric AML patients compared to normal controls. Fifty-one mRNA levels were significantly upregulated, while 85 mRNA levels were significantly downregulated by >10-fold in pediatric AML, compared to normal controls. GO terms and KEGG pathway annotation data revealed that cell cycle pathway-related genes were significantly associated with pediatric AML. As confirmed by qRT-PCR, expression of 24 of 97 lncRNA was altered in pediatric AML compared to normal controls. In pediatric AML, ENST00000435695 was the most upregulated lncRNA, while ENST00000415964 was the most downregulated lncRNA. Data from this study revealed dysregulated lncRNAs and mRNAs in pediatric AML versus normal controls that could form gene pathways to regulate cell cycle progression and immunoresponse. Further studies are required to determine whether these lncRNAs could serve as novel therapeutic targets and bbdiagnostic biomarkers in pediatric AML.

Determination of ETV6-RUNX1 genomic breakpoint by next-generation sequencing.

The t(12;21)(p13;q22) ETV6-RUNX1 gene fusion is one of the most common chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). It is associated with favorable prognosis. The identification of the genomic sequence of the breakpoint flanking regions of the ETV6-RUNX1 translocation should be the best strategy to monitor minimal residual disease (MRD) in patients with ETV6-RUNX1-positive ALL. In this study, the ETV6-RUNX1 translocation was sequenced by next-generation sequencing (NGS) in 26 patients with ETV6-RUNX1-positive ALL and re-sequenced by using the Sanger method. Interestingly, the three-way translocation, including ETV6-RUNX1, was detected in five patients. Four of them relapsed during or after therapy, while 21 patients without the three-way translocation were still in remission (P < 0.0001). The three-way translocation pattern was identical between the diagnosis and relapse samples in three patients, excluding one patient (SCMC-001245). The relapse samples retained the translocation of ETV6-RUNX1 relative to the three-way translocation t(8;12;21) at diagnosis, suggesting that the three-way translocation might be an important risk factor for relapse in patients with ETV6-RUNX1-positive ALL and should be further studied.

Clinical Features and Treatment Outcomes of Childhood Autoimmune Hemolytic Anemia: A Retrospective Analysis of 68 Cases.

Autoimmune hemolytic anemia (AIHA) is a rare disease in children, and its clinical severity varies. To better understand disease manifestation and treatment outcome, we analyzed 68 children diagnosed as AIHA for clinical characteristics, laboratory findings, and treatment outcomes. Data show that primary AIHA accounted for 39.7% of all patients, whereas secondary AIHA accounted for 60.3%. Among them, Evans syndrome (ES) accounted for 20 cases (29.4%). Average hemoglobin was lower in the 1-year or below age group than in the above 1-year age group, combined-antibody group than single-antibody group, and IgM-contained group than non-IgM-contained group (P<0.05 for all). The duration of therapy in the ES group was longer than that in the AIHA-only group (P<0.05). During the follow-up period, 29 cases (29/45, 64.4%) remained in continuous remission. In total, 35.6% of patients relapsed after first complete remission and 56.3% of them still showed good response to glucocorticoid after relapse. There was no difference in the duration of therapy or relapse rate between the intravenous immunoglobulin (IVIG)-treatment group and the non-IVIG-treatment group. In conclusion, the severity of anemia correlates with age and serologic types of direct antiglobulin test. Glucocorticoid is efficacious for AIHA regardless of whether it is a first attack or relapse in this cohort of young patients. ES needs longer treatment duration. IVIG does not improve the outcome of AIHA.

[Efficacy Analysis of Allogeneic Hematopoietic Stem Cell Transplantation for Children with Severe Aplastic Anemia].

OBJECTIVE: To study the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children with severe aplastic anemia (SAA). METHODS: A total of 11 children with SAA were treated with HLA matched siblings (n = 7), umbilical cord blood (n = 2) and haploidentical HSCT (n = 2). RESULTS: Among 11 children patients, 10 patients achieved engraftment, but 3 children patients experienced secondary graft failure, after donor lymphocyte infusions (DLI), they achieved engraftment again. One patient received cord blood transplantation and experienced primary graft rejection, but acquired autologous recovery. The median time for neutrophils to reach over 0.5 × 10(9)/L was 14 days (10-19 days) in the 9 children received bone marrow or bone marrow and peripheral blood allo-HSCT, while the median time for platelets to reach over 20 × 10(9)/L was 17 days (8-42 days). For the patient received double cord blood transplantation, the time of neutrophile and platelet level recovery was 16 days and 41 days, respectively. CONCLUSION: If HLA-matched sibling donor is available, allo-HSCT can be recommended as the first line of treatment for children with SAA. It is feasible for children with SAA to receive allo-HSCT from selective donor, including cord blood and haploidentical HSCT. Donor lymphocyte infusions can improve engraftment.

Clinical features and prognostic significance of TCF3-PBX1 fusion gene in Chinese children with acute lymphoblastic leukemia by using a modified ALL-BFM-95 protocol.

For children with precursor B (pre-B) acute lymphoblastic leukemia (ALL) with TCF3-PBX1 fusion gene, their prognosis has been a controversial topic. From January 2008 to December 2012 in our hospital, 450 patients were diagnosed as ALL. Clinical characteristics of 20 patients with TCF3-PBX1 fusion gene were analyzed retrospectively, which were classified to the intermediate-risk (IR) group according to Chinese Children Leukemia Group-2008 (CCLG-2008) risk-stratification criteria and protocol based on the backbone of BFM 95 trails. Eighty five cases without TCF3-PBX1 in the same IR group were regarded as the comparison group. There were no differences in age, gender, initial white blood cell (WBC) count, status of central nerves system (CNS) at diagnosis and complete remission (CR) rates of bone marrow (BM) between the two groups (P > .05). The 5-year probability of event-free survival (EFS) rates were 84.4 ± 15.6% and 73.5 ± 15.6% in the TCF3-PBX1 group and the comparison group (P = .35), respectively. The 5-year probability of overall survival (OS) rates were 86.0 ± 17.6% and 81.8 ± 17.6% (P = .46), respectively. Relapse rates were 10.5% and 12.9% (P = 1.00), respectively. There were not cases with CNS relapse in the TCF3-PBX1 group. When intensive chemotherapy was used, the TCF3-PBX1 was associated with a favorable outcome in childhood pre-B ALL.

[Effect of DNA methylation on the proliferation of leukemia cells regulated by miR-34b].

OBJECTIVE: To study the expression level and CpG island methylation status of miR-34b in leukemia cell lines and to research the effect of DNA methylation on the proliferation of leukemia cells regulated by miR-34b. METHOD: Taqman real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was carried out to detect the relative expression of miR-34b in control group (bone marrow cells of 20 children without blood disease) and 8 leukemia cell lines (U937, HL-60, MV4-11, M2R, K562, Raji, CCRF, DAMI). Methylation-specific polymerase chain reaction (MSP) was carried out to detect the methylation differences of miR-34b in control group (Bone marrow cells of 23 children without blood disease), 8 leukemia cell lines. HL-60 and K562 were treated with methyltransferase inhibitor (5-aza-2-dC) for further detection of its methylation status and expression of miR-34b. Hsa-miR-34b mimics was transfected into K562 cell by liposome, the transfection efficiency was detected by flow cytometry. The cell proliferation of hsa-miR-34b transfected group in each stage was measured with CCK-8 assay, and then compared with non-transfected group and negative control group. RESULT: The relative expression level of miR-34b in the group of children without blood disease and the group of leukemia cell lines were 5.22 ± 1.15, 0.03 ± 0.03. The results showed that, the group of leukemia cell lines was significantly different from the control group (t = 4.538, P < 0.01) . Eight leukemia cell lines showed methylation, the positive rate of the methyl was 100%. There was no methylation in the 23 cases of control group. After leukemia cell lines HL-60 and K562 were treated with 5-aza-2-dC, the methylated bands became obviously weakened, and the relative expression levels of miR-34b substantially increased 49.5 times and 18.8 times respectively. After hsa-miR-34b mimics was transfected into K562 cell by liposome, its transfection efficiency detected by flow cytometry was 61% and the cell proliferation was measured with CCK-8 assay from which it was found that the cell proliferation was significantly suppressed compared with the control group at 48 h (t = 9.303, P < 0.01), 72 h (t = 65.617, P < 0.01), 96 h (t = 36.878, P < 0.01) and 120 h (t = 18.748, P < 0.01) in hsa-miR-34b transfected group, with the inhibition rate of 12.2% (48 h), 45.7% (72 h), 32.5% (96 h) and 22.9% (120 h). CONCLUSION: The hypermethylation of promoter leads to decrease in the expression levels of miR-34b in leukemia cell lines, which attenuate mechanism of proliferative inhibition may be one of the reasons of occurrence or development of childhood leukemia.

[Clinical significance of glucocorticoid induction test in Chinese childhood acute lymphoblastic leukemia].

OBJECTIVE: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, while glucocorticoid (GC) is a critical component in multi-agent chemotherapy protocols currently used for the treatment of ALL. The purpose of this study was to investigate the relationship between the glucocorticoid induction test and the clinical features and the prognosis of Chinese childhood ALL. METHOD: The study recruited 309 hospitalized patients (187 male and 122 female) with childhood ALL, the sex, age, initial WBC count, immunophenotype, chromosome and gene expression were recorded. After diagnosis, all patients received GC induction test for 7 days. Then they were divided into prednisone good response (PGR) group and prednisone poor response (PPR) group according to the peripheral lymphoblast count on D8. Early responses to chemotherapy and treatment outcomes of the patients in the two groups were also analyzed. RESULT: Of the 309 patients, 263 belonged to PGR group and 46 belonged to PPR group. Initial WBC count was higher in PPR group than in PGR group (86.30×10(9)/L vs. 30.97×10(9)/L, P < 0.01) . B lineage ALL showed more sensitive to GC than T-ALL (86.6% vs. 60%, P < 0.05). Different initial-risk-group's sensitivity to GC differed from one another (high-risk:51.4%, medium-risk: 82.7%, standard risk: 93.7%, P < 0.0125). There was no significant difference between two groups in chromosomal karyotypes (P > 0.05). BCR-ABL positive ALL showed lower sensitivity to GC (P < 0.05) , while MLL, TEL-AML1, E2A-PBX1 positive rates in two groups were of no statistical significance (P > 0.05). Bone marrow was reviewed on D15 and D33, and the CR rates in PGR group were significantly higher than that in PPR group (D15: 60.5% vs. 32.6%, D33: 94.6% vs. 73.3%, P < 0.01) ; Minimal residual disease (MRD) levels were examined on D33, W12, and both were much lower in PGR group (D33: P < 0.01, W12: P < 0.05). Of the PGR group 215 patients (81.7%) remained continuously in complete remission (CCR) while only 28 cases (60.9%) in PPR group did so. The CCR rate was much higher in PGR group than that in PPR group (P < 0.01). CONCLUSION: Closely related to clinical features and the outcomes of treatment, GC induction test is also an important prognostic factor in Chinese childhood ALL.

[The expression and clinical significance of miR-203 in pediatric acute leukemia].

OBJECTIVE: To investigate the methylation, expression and clinical significance of miR-203 in pediatric acute leukemia. METHODS: The methylation status of miR-203 promoter CpG islands was detected with methylation-specific polymerase chain reaction. The expression of miR-203 was detected by Taqman real- time quantitative polymerase chain reaction. And the clinical significance of miR-203 in pediatric acute leukemia (ALL) was also analyzed. RESULTS: The promoter of miR-203 was unmethylated in all of 31 pediatric acute lymphoblastic leukemia, all of 15 pediatric acute myeloid leukemia (AML) and all of 23 controls. The relative expression levels of miR-203 in controls, pediatric acute leukemia, ALL and AML were 16.93±6.31, 48.97±10.38, 55.88±12.91, 24.28±9.10 respectively. The results indicated that miR-203 was significantly up- regulated in pediatric acute leukemia (P=0.011) and ALL (P=0.009), not in pediatric AML (P=0.514) compared with control. The expression of miR-203 was significantly related with the gender, immunophenotype, chromosome, fusion gene, BCR-ABL, SIL-TAL1 and prednisone experiment in pediatric ALL and the gender, chromosome, fusion gene, SIL-TAL1 in pediatric acute leukemia (P<0.05). And in risk stratification pairwise comparisons, the expression of miR-203 in the medium-risk and high-risk groups appeared significantly different (P=0.022). CONCLUSION: miR-203 may not be regulated with methylation mechanism in pediatric acute leukemia. miR- 203 may be a protooncogene involved in the formation of pediatric acute leukemia and ALL. Further analyses indicated that high expression of miR-203 may be associated with poor prognosis of pediatric ALL and acute leukemia.

[The effect of the adverse events with thiopurine S-methyltransferase gene mutation on outcome of childhood acute lymphoblastic leukemia].

OBJECTIVE: To investigate thiopurine S-methyltransferase (TPMT) activity and gene promoter polymorphism to probe its significance of individual chemotherapy in acute lymphoblastic leukemia (ALL) children. METHODS: HPLC method was carried out to determine TPMT activity (n=100), which activity at newly diagnosed. At the same time determination of TPMT activity in healthy children (n=180), these children come from the health care clinic. Using online primer3 software design primers, PCR products were purified. To sequence TPMT gene of the patients with clinical events(n=30). According to the method to analysis of correlation between TPMT activity and toxicity. RESULTS: The average TPMT activities were (31.72±10.31) nmol·g⁻¹Hb·h⁻¹ and (30.70±9.67) nmol·g⁻¹Hb·h⁻¹ in ALL and healthy groups respectively, without gender differences of TPMT activities (P=0.45) in both groups. The TPMT activity with clinical events in newly diagnosed ALL patients (n=30) was (24.07±11.43) nmol·g⁻¹Hb·h⁻¹. There are significant differences of TPMT activities between severe bone marrow suppression [(20.96±7.24) nmol·g⁻¹Hb·h⁻¹] and ALL patients with clinical events groups (P<0.05). The TPMT activity of (40.46±8.18) nmol·g⁻¹Hb·h⁻¹ in recurrence children was also significantly different (P<0.05). TPMT activity in severe liver toxicity group was not significantly different (P=0. 930). Of TPMT gene sequencing in ALL patients with clinical events, only 3 children were heterozygosity mutations of TPMT*3C, while others homozygous genotype. There were significant differences of TPMT activities between heterozygosity genotype [(11.99±1.32) nmol·g⁻¹Hb·h⁻¹] and homozygous genotype groups [(24.95±11.32) nmol·g⁻¹Hb·h⁻¹] (P<0.05). There were five kinds of variations at the vicinity of the promoter region of -100 of tandem repeats (VNTR) polymorphism（*V3/*V3、*V3/*V4、*V4/*V4、*V5/*V5、*V4/*V6）without significant differences of TPMT activities among five kinds (P=0.186). CONCLUSION: TPMT activity was related to the gene polymorphism. TPMT activity determination had prognostic value and guided individualized treatment.

[Clinical significance of dynamic monitoring the minimal residual disease in childhood B-lineage acute lymphoblastic leukemia by multiparameter flow cytometry].

This study was aimed to explore the clinical significance of monitoring level of minimal residual disease (MRD) at different time point in B-lineage childhood acute lymphoblastic leukemia (B-ALL). Two hundred and six children with B-ALL were enrolled in this study from Augest 2008 to September 2011 in our hospital. MRD levels were detected by flow cytometry at day 15, 33 and week 12 after initial chemotherapy. The event-free survival (EFS) for patients based on MRD levels measured at different stages of chemotherapy were compared by Kaplan Meier analyses. The results showed that out of 206 cases 196 cases achieved complete remission (CR) after induction therapy (CR rate 95.1%), the 1- and 3-year EFS rate were (92.7 ± 1.8)% and (78.7 ± 3.7)%, respectively, and the 3-year EFS rate was (85.6 ± 4.9)% in standard risk group, (82.1 ± 5.8)% in intermediate risk group and (58.1 ± 9.2)% in high risk group, there was significant statistical difference between above mentioned 3 groups (P < 0.001). The MRD analysis at different time points showed that the higher the MRD level, the lower the 3-year EFS rate of children with ALL, in which the 3-year EFS rate of MRD ≥ 10(-2) at day 15, MRD ≥ 10(-3) at day 33 and MRD ≥ 10(-3) at week 12 were significantly lower. The MRD ≥ 10(-3) at week 12 was proven to be an independent predictor by multivariate Cox proportional-hazards regression model. The 3-year EFS rate for patients with MRD < 10(-3) and MRD ≥ 10(-3) at week 12 were (86.3 ± 4.1)% vs (55.8 ± 9.1)% (P < 0.05); 8 relapsed among 98 cases with negative MRD (MRD < 10(-4)) at day 33, 19 relapsed among 108 cases with positive MRD at day 33 between the two groups for recurrence rate has significant difference (P < 0.05). It is concluded that dynamically monitoring MRD by multi-parameter flow cytometry can precisely evaluate treatment response, judge treatment outcome and predict relapse in childhood B-ALL. The MRD 10(-2) at day 15, MRD 10(-3) at day 33 and MRD 10(-3) at week 12 should be considered as the best cut-off. MRD ≥ 10(-3) at week 12 was proven to be an independent factor of poor prognosis.

[Relationship between genetic polymorphism of multidrug resistance 1 gene and the risk of childhood acute lymphocytic leukemia].

OBJECTIVE: To investigate the relationship between genetic polymorphism in exon 12 C1236T, exon 21 G2677T/A and exon 26 C3435T of the multidrug resistance 1 (MDR1) gene and the risk of childhood acute lymphocytic leukemia (ALL). METHOD: A total of 176 patients with ALL and a cohort of 170 matched healthy subjects were included. SNaPshot SNP typing was used to determine the genotypes of MDR1 C1236T, G2677T/A, C3435T. Based on the clinical data, the relationship between genetic polymorphism of MDR1 and the risk of childhood ALL was analyzed. RESULT: There was significant difference in the distribution of genotype of MDR1 C3435T between the group of controls and cases. The mutant homozygous TT genotype was found to be associated with occurrence of ALL (P = 0.000; OR = 4.504). The data show evidence of pairwise linkage disequilibrium between the three common SNPs (C1236T-G2677T/A-C3435T). The haplotypes of TTT, TGC, CGC and CAC were predominant. The haplotype CGT distributed significantly differently between the groups of controls and cases (P = 0.034). The frequency of the haplotype TTT/TTT in the high risk group was higher than the other groups (P = 0.037). CONCLUSION: The present findings suggest that 3435C→T polymorphism in MDR1 gene may be a genetic susceptibility factor for ALL. The haplotype of MDR1 (C1236T-G2677T/A-C3435T) could be the clinical parameter at diagnosis.