Clinical significance of CASP8AP2 gene methylation in childhood acute lymphoblastic leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the methylation level in the promoter of caspase 8 associated protein 2 (CASP8AP2) gene between samples at diagnosis and in complete remission, and to investigate its relationship with clinical features and prognosis in children with acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>Diagnostic DNA samples from 109 newly diagnosed children with ALL admitted from August 2007 to March 2010, and 94 ALL children in CR (complete remission) among them were collected. Bisulfite modification and MethyLight method established by our research team were used to determine the methylation level of the two key CpG sites (at -1189 and -1176) of the promoter of CASP8AP2 gene.</p><p><b>RESULTS</b>The average methylation level of the two CpG sites in newly diagnosted samples was higher than that in CR samples (71.1% ± 1.7% vs 64.2% ± 21.2%) (P = 0.008). Analysis with receiver operating characteristic (ROC) curve showed that the area under curve was 0.687 (P = 0.024), indicating that the methylation level of the two CpG sites was able to predict relapse efficiently to some extent, 76.9% was chosed as a cutoff value to divide the patients into high methylation group (49 patients) and low methylation group (60 patients). The incidence of relapse in high methylation group was higher than that in low methylation group (20.4% vs 6.7%) (P = 0.044), five year relapse free survival in high methylation group was also lower than that in low methylation group (Log rank, P = 0.033). Furthermore, high methylation at new diagnosis were correlated with high level of minimal residual disease (MRD) before consolidation therapy (P = 0.011). In the 34 children with MRD ≥ 10(-4) at the end of induction remission, the relapse rate of high methylation patients was significantly higher than that of low methylation patients (8/16 vs 3/18)(P = 0.038).</p><p><b>CONCLUSION</b>The abnormal hypermethylation of the two CpG sites (at -1189 and -1176) of the promoter of the CASP8AP2 gene is possibly associated with leukemogenesis in childhood ALL. The treatment outcome is more poor in patients with hypermethylation than that in patients with low methylation. The combination of the methylation level of the two CpG sites and MRD level at the end induction remission is able to predict relapse more effectively.</p>

Correlation analysis of FPGS rs10760502G>a polymorphism with prognosis and MTX-related toxicity in pediatric B-cell acute lymphoblastic leukemia / 中国实验血液学杂志

This study was aimed to explore the relation between folylpolyglutamate synthetase (FPGS) rs10760502 polymorphism and prognosis and methotrexate (MTX)-related toxicities in pediatric B-cell acute lymphoblastic leukemia (B-ALL). Sequenom MassARRAY was used to genotype rs10760502. The χ(2) test, Kaplan-Meier method and Cox regression models were used to analyze the data. The results indicated that A allele carriers (GA+AA) had poor relapse free survival (RFS, log-rank: P = 0.004) and event free survival (EFS, log-rank: P = 0.022) compared with the GG genotype carriers. Multivariate Cox-regression analysis results showed that A allele is an independent prognosis factor for poor RFS [hazard ratio (HR), 20.173; 95% CI, 2.535-160.545; P = 0.005] and EFS (HR, 8.133; 95% CI, 1.718-38.512; P = 0.008). No relationship was found between any MTX toxicity and rs10760502 polymorphism. It is concluded that FPGS rs10760502G>A polymorphism may affect the treatment outcome of B-ALL patients.

Evaluation of early response to treatment and its prognostic value in childhood acute lymphoblastic leukemia / 中国实验血液学杂志

This study was purposed to investigate the prognostic value of early response to treatment in childhood acute lymphoblastic leukemia (ALL). Four indexes were used to assess early response to treatment including response to prednisone on day 8 (D8-PR), percentage of lymphoblast in bone marrow on day 22 (D22-BM) and day 33 (D33-BM), the level of minimal residual disease (MRD) on day 33 (D33-MRD) by morphological and molecular biological method in 426 children with ALL. Prognostic impact of early response to treatment was analyzed, and multivariate analysis of the predictive value was performed by Cox-regression analysis. All patients were followed up until October 31, 2013, with a median follow-up time of 80 months (0.5 to 106 months). The results showed that there were significant differences between event free survivals (EFS) of the sub-groups divided according to the four indexes. The 8 years-EFS in patients with prednisone good response (PGR) was significantly higher than that in patients with prednisone poor response (PPR);patients with M1 in bone marrow on day 22 or day 33 had the better outcomes than that of patients with M2/M3;patients with high level of MRD ( ≥ 10(-4)) had the worse outcomes as compared with patients with low level of MRD (<10(-4)) (P < 0.001). Cox proportional hazard model analysis showed that BCR/ABL fusion gene positive, D8-PR, D33-BM and D33-MRD were the independent prognostic factors for childhood ALL, and the hazard ratio of D33-MRD ≥ 10(-2) was highest (HR:11.886, P < 0.001). It is concluded that early response to treatment is an independent prognostic factor with important prognostic values, and it has important clinical guiding instructive significance for risk stratification in the treatment of children ALL.

Evaluation of the efficacy of two successive protocols on pediatric acute lymphoblastic leukemia with E2A-PBX1 fusion gene / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of BCH-03 and CCLG-08 protocols in treating E2A-PBX1 pediatric acute lymphoblastic leukemia (ALL).</p><p><b>METHOD</b>From January 2003 to January 2011, 59 ALL patients identified as E2A-PBX1 were analyzed in a retrospective study. There were 37 and 22 patients treated with Protocol BCH-03 and CCLG-08, respectively. The clinical characteristics at diagnosis, response to early treatment, the time of relapse, relapse-free survival (RFS) and event-free survival (EFS) in the two groups were analyzed.</p><p><b>RESULT</b>There were no significant differences in gender, age, initial white blood cell count, the central nervous system involvement, immunophenotype, prednisone response, the rate of complete remission, and the time of relapse between the two groups (P > 0.05). The only difference in induction therapy of the two protocols existed in the glucocorticoids used, that is, BCH-03 used 60 mg/m(2) prednisolone and CCLG-08 used 6 mg/m(2) dexamethasone. The doses of vincristine, daunorubicin and L-asparaginase were the same in the two groups. At the end of induction therapy, the MRD negativity rate in BCH-03 group was significantly higher than that in CCLG-08 group (84.2% vs. 47.1%, P = 0.018). The incidences of severe infection of the two groups during induction of remission were similar (P = 0.135). The EFS of BCH-03 group was significantly superior to that of CCLG-08 group (94.5% vs. 71.5%, P = 0.010), and the RFS of BCH-03 group tended to be better than that of CCLG-08 group (94.5% vs. 78.6%, P = 0.059).</p><p><b>CONCLUSION</b>Compared to Protocol CCLG-08, Protocol BCH-03 was more effective for pediatric E2A-PBX1 ALL, and 60 mg/m(2) prednisolone was more suitable for the induction therapy of this subtype of pediatric ALL.</p>

Suppression of NAMPT expression enhances the sensitivity of K562 cells to imatinib and its relative mechanism / 中国实验血液学杂志

The aim of this study was to investigate the effect of suppression of nicotinamide phosphoribosyltransferase (NAMPT) expression on imatinib-sensitivity in chronic myelogenous leukemia (CML) cell line K562 and its mechanisms, NAMPT siRNA was synthesized and transfected into K562 cells. PI/Calcein staining technique was used to determine survival rate of transfected K562 cells at 48th hour after exposure to 1 µmol/L imatinib. MTS method was used to determine the proliferation changes of transfected K562 cell at 48th hour after exposure to different doses of imatinib, then half inhibitory concentration (IC(50)) was calculated. Expression of NAMPT at 3rd-48th hour after exposure to 1 µmol/L imatinib was determined by Western blot. To explore the effect of NAMPT-siRNA and imatinib on the expression of apoptosis-related genes, the microarray data from NCBI GEO Data-Sets was analyzed, then the results were confirmed by Western blot. The luciferase reporter assay was used to determine the effect of NAMPT and imatinib on transcriptional activity of NF-κB transcription factors. The results showed that after exposure to 1 µmol/L imatinib for 3 - 48 h, there was no significant change of NAMPT expression in K562 cells. The expression of NAMPT could be effectively inhibited by the NAMPT-siRNA. After exposure to 1 µmol/L of imatinib for 48 h, the survival rate of NAMPT-siRNA interference group was lower than that of negative control group (P < 0.05), indicating that suppression of NAMPT expression can increase the sensitivity of K562 cells to imatinib and enhance the killing effect of imatinib on K562 cells. The IC(50) of imatinib in NAMPT-siRNA interference group was the lowest compared with that of control group (P < 0.05) after exposure to different concentrations of imatinib for 48 h, the fitted survival curves showed that the slope of NAMPT-siRNA interference group was the largest ranging between 0.01 - 0.1 µmol/L of imatinib. Data mining of expression profiling indicated that the anti-apoptotic factor Bcl-2 decreased in K562 cells treated with either NAMPT-siRNA or imatinib, which was confirmed by Western blot. The inhibitory effect was much more significant when both NAMPT-siRNA and imatinib were used. The results of luciferase reporter assay showed that either NAMPT-siRNA or imatinib decreased transcriptional activity of NF-κB. The decreased effect was much more significant when both NAMPT-siRNA and imatinib were used. It is concluded that survival of K562 cells affected by imatinib may not be due to regulation of expression of NAMPT. When expression of NAMPT decreases, the K562 cells are more sensitive to imatinib, this may be related with the decreased transcriptional activity of NF-κB and its downstream effector Bcl-2.

Thiopurine S-methyltransferase gene sequence analysis of ALL children severely intolerant to 6-mercaptopurine / 中国实验血液学杂志

This study was aimed to analyze the thiopurine S-methyltransferase (TPMT) gene sequence in acute lymphoblastic leukemia (ALL) children severely intolerant to 6-mercaptopurine (6-MP) and to investigate the causes resulting in tolerance difference to 6-MP in ALL children so as to provide evidence for safe and rational use of 6-MP. The adverse reactions of drug was evaluated in ALL children treated with BCH-2003-ALL chemotherapeutic protocol during 2004-10-1 to 2007-9-30 according to NCI-CTC V2.0. The TPMT gene sequences of ALL children with 3-4 grade of severe toxicity during the maintenance therapy were analyzed by PCR and direct DNA sequencing. To assure the accuracy of sequencing, the 738 bp fragment of coding region in TPMT gene (NM_000367) was divided into 3 subfragments and bidirectionally sequenced. The results indicated that among 133 ALL children, 61 were severely intolerant to 6-MP. The direct DNA sequencing showed that among 59 patients (excluding 2 cases without RNA samples), the simple myelotoxicity was found in 37 cases, hepato-myelotoxicity was observed in 9 cases, hepatotoxicity along appeared in 12 cases, 1 case showed skin rash. Out of 59 ALL children, the C474T mutation was found in 57 cases, with mutation rate 96.6%, including 21 cases with heterozygous mutation and 36 cases with homozygosis mutation. The TPMT gene sequencing of 10 cases tolerant to 6-MP indicated that C474T mutation was detected in 8 cases which was homozygous mutation. It is concluded that the C474T mutation in 738 bp fragment of coding region in TPMT gene is very frequent, but it is not related with tolerance to 6-MP, suggesting that severe intolerance to 6-MP in ALL children may be not related with the mutation of coding region in TPMT gene.

Gene rearrangement pattern of immunoglobulin and T-cell receptor (Ig/TR) and its clinical characteristics in children with SET-NUP214 fusion gene-positive leukemia/lymphoma / 中国实验血液学杂志

The purpose of this study was to analyze the gene rearrangement pattern of immunoglobulin and T-cell receptor (Ig/TR) and its clinical characteristics in three children with SET-NUP214 fusion gene positive leukemia/lymphoma. The transcript of SET-NUP214 fusion gene was detected by RT-nested PCR. The pattern of Ig/TR gene rearrangement was analyzed by using the BIOMED-2 multiplex PCR assays. Allelic-specific primers were designed for further monitoring the minimal residual disease (MRD). The results indicated that the fusion site located between exon 7 of SET and exon 18 of NUP214 at mRNA level in the three patients. The diagnoses were made as the mixed phenotype of acute leukemia (MPAL) for patients 1, acute T-lymphoblastic leukemia (T-ALL) for patients 2, and stage IV T-lymphoblastic lymphoma (T-LBL) for patients 3, respectively. Patient 1 responded to chemotherapy very poorly and relapsed at month 6 after hematopoietic stem cell transplantation. Patient 2 had high MRD (> 10(-2)) at the end of inducing remission therapy (day 33) which implied poor outcome, and died of toxic epidermal necrolysis and sequent serious infection. Patient 3 achieved hematological complete remission (CR) and MRD negative at day 15 and day 33 respectively. The duration of CR lasted for 30 months. Clonal TR gene rearrangements were detected in all the three patients. The rearrangements of TRD, TRG and TRB were found in patient 1 and 3. The rearrangements of TRD, TRB, IgH and IgK Kde were detected in patient 2. All the 6 TRB rearrangements detected were incomplete rearrangements, whereas 85.7% and 14.3% of the TRD, and TRG rearrangements were complete and incomplete, respectively. It is concluded that the transformation of SET-NUP214(+) leukemia/lymphoma cells may occur after the rearrangements of TRD and TRG and shortly after TRB rearrangement. The leukemia/lymphoma cells of patient 1 and 2 are more immature which may be related with poor outcome or response to chemotherapy.

Characteristics of fusion gene and immunophenotype in MLL gene rearrangement positive childhood acute lymphoblastic leukemia / 中国实验血液学杂志

The study was aimed to investigate the fusion gene transcript and immunophenotypic characteristics of the mixed linage leukemia (MLL)-rearranged positive childhood acute lymphoblastic leukemia (ALL). The incidence of MLL rearrangement in 601 cases of ALL patients was detected by the multiple-nested polymerase chain reaction (PCR); the subtypes and features of the fusion gene transcript were analyzed by PCR products sequencing; the immunophenotypic characteristics at diagnosis were compared between the 22 MLL rearrangement positive of ALL patient, 30 negative control which selected randomly from the patients whose fusion gene could not be detected in the same term and 43 pro-B-ALL patients. The results showed that the incidence of MLL positive ALL was 3.66%, constituted 29.9% of the pro-B-ALL. The MLL rearrangement positive 20 B-ALL patients were all CD10 negative; the number of patients who carried CD13, CD33 and CD34 was lower than that of pro-B-ALL who had no fusion gene, whereas the expression of CD20, CD22, CD2, CD5, CD7 showed no difference. 4 kind partner genes of MLL-AF4, AF9, AF10 and ENL were detected. The fusion loci of MLL gene were mainly located at the exon 6, 7, 8 and many kind of fusion loci of MLL may exist in one patient; whereas its partner gene fusion loci were relatively single. A transcript contains a random insert sequence existed in a transcript of one MLL-AF10+ patient. It is concluded that though incidence of MLL rearrangement is low, but it has a variety of fusion transcripts, the ALL patients has unique biological characteristics at immunophenotype and fusion transcript.