Efficacy of Chimeric Antigen Receptor T Cell in the Treatment of Refractory/Recurrent B Acute Lymphocytic Leukemia in Children / 中国实验血液学杂志

OBJECTIVE@#To observe the efficacy of chimeric antigen receptor T cell (CAR-T) in the treatment of children with refractory/recurrent B acute lymphocytic leukemia (B-ALL).@*METHODS@#Thirty-two patients with r/r B-ALL were treated by CAR-T, the recurrence and death respectively were the end point events to evaluate the efficacy and safety of CAR-T.@*RESULTS@#The median age of the patients was 7.5 (2-17.5) years old; 40 times CAR-T were received in all patients and the median number of CAR-T was 0.9×107/kg; efficacy evaluation showed that 2 cases died before the first evaluation. Thirty patients showed that 3, 6, and 9-moth RFS was (96.3±3.6)%, (81.4±8.6)% and (65.3±12.5)%, respectively, while 3, 6, and 9-month OS was all 100%, and 12, 24-month OS was (94.7±5.1)% and (76±12.8)%. BM blasts≥36% before reinfusion and ferritin peak≥2 500 ng/ml within two weeks of CAR-T cell reinfusion were associated with recurrence. Adverse reactions mainly included cytokine release syndrome (CRS) and CART-cell-related encephalopathy syndrome (CRES), CRS appeared in 26 patients within a week of CAR-T cell reinfusion. CRES reaction was detected in 12 patients. Eighteen patients received intravenous drip of tocilizumab, among them, 12 combined with glucocorticoid. CRS and CRES reactions were relieved within one week after treatment. Hormone dosage was related to the duration of remission in patients, and the cumulative dose of methylprednisolone≥8 mg/kg showed a poor prognosis.@*CONCLUSION@#CAR-T is a safe and effective treatment for r/r B-ALL, most CRS and CRES reactions are reversible. BM blasts ≥36% before reinfusion and cumulative dose of methylprednisolone ≥8 mg/kg after reinfusion both affect the therapeutic effect. Ferritin≥2 500 ng/ml within two weeks after reinfusion is related to disease recurrence and is an independent prognostic risk factor.

Long-term outcomes of childhood lymphoblastic lymphoma: report of 70 cases / 中华血液学杂志

<p><b>OBJECTIVE</b>To summarize long-term outcomes of childhood lymphoblastic lymphoma (LBL) with protocol CCCG-97 and -2002.</p><p><b>METHODS</b>From November 1998 to October 2010, 70 consecutive newly diagnosed childhood LBL (5 B-LBL and 65 T-LBL) were enrolled in this study, in which 22 received CCCG-97 and 48 CCCG-2002 protocols. St.Jude staging system was adopted. Patients were divided into three risk groups based on clinical stage and serum LDH, and received chemotherapy with different intensity. The factors, which were possibly associated with the prognosis, were analyzed. The survival rates were evaluated by Kaplan-Meier analysis.</p><p><b>RESULTS</b>The patients were 1.5 to 14 years old with the median age of 8 years old. They were evaluated as stage I-II for 6 , stage III41, and stage IV23 (15 were BM positive and 8 multiple bone metastases). Until Dec.31th, 2011,the mean follow-up was 62.5 months (range, 14 to 161 months) with the median follow-up of 48 months. 1-year overall survival (OS) was 74.3%, and 5- year event-free survival (EFS) 64.1% (abundance as event). Thirteen patients were complicated with serious condition during chemotherapy and 1 died of complication. Univariate analysis indicated that delayed and/or non-completed response on days 33 and 63 of induction was the unfavorable prognostic factor.</p><p><b>CONCLUSION</b>Primary LBL usually located in the mediastinum. 90% of the patients was at advanced stage III-IV at first presentation. The 5-year EFS was 64.1%. Patients not achieved CR at days 33 and 63 at the end of induction was a poor prognostic factor.</p>

Expression of zinc finger protein X-linked in childhood B lineage acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the expression of zinc finger protein X-linked (ZFX) in bone marrow mononuclear cells (BMMCs) of children with B lineage acute lymphoblastic leukemia (B-ALL) and its relationship with prognosis.</p><p><b>METHODS</b>The expression of ZFX in human leukemia cell lines (REH, HL-60, NB(4) and K562) was measured by Western blot. ZFX gene was cloned by PCR from one patient and DNA sequencing technology was used to confirm it. Real-time PCR was used for detecting ZFX mRNA expression in the BMMCs of 82 children with newly-diagnosed B-ALL, 24 children with complete remission (CR) after induction therapy and 64 control children (fracture or congenital heart disease patients). According to the presence of bone marrow or central nervous system relapse during a follow-up of 3 years, the patients were identified as having a good or poor prognosis. Their ZFX mRNA levels in BMMCs at diagnosis were compared.</p><p><b>RESULTS</b>ZFX protein was expressed in human leukemia cell lines REH, HL-60, NB(4) and K562. ZFX mRNA expression was significantly higher in the newly-diagnosed ALL group than in the control group (P < 0.01). ZFX mRNA expression in the ALL CR group was significantly reduced compared with the newly-diagnosed ALL group (P < 0.01). Children with a poor prognosis had significantly higher ZFX mRNA levels at diagnosis than those with a good prognosis (P < 0.05).</p><p><b>CONCLUSIONS</b>ZFX is over-expressed in children with B-ALL and its levels are higher in those with a poor prognosis than those with a good prognosis, which suggests that ZFX is important in the prognosis evaluation of B-ALL.</p>

The abnormal expression of IKZF1 encoded protein-IKAROS in B-ALL children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To analyze the isoforms of IKAROS in the bone marrow samples from children with acute B-lineage lymphoblastic leukemia (B-ALL) and to investigate the relationship between frequency of dominant-negative (DN) IKAROS isoforms and prognosis of B-ALL, and to preliminarily study the relevant mechanism.</p><p><b>METHODS</b>A total of 137 children with newly diagnosed B-ALL, who sequentially entered the Department of Hematology and Oncology, Shanghai Children's Medical Center between January 2005 and September 2010, were included in the study. Nest-PCR, Sanger sequencing, and TA cloning were used to analyze the expression of IKAROS isoforms in these children. The relationship between frequency of DN IKAROS isoforms and prognosis of B-ALL was investigated.</p><p><b>RESULTS</b>Of the 137 children with newly diagnosed B-ALL, 16 had expression of IK6, 14 had expression of IK4, and 2 had expression of IK7. There was significant difference in 2.5-year event-free survival between the cohorts of DN IKAROS and non-DN IKAROS (P=0.01). Analysis of the 10 paired of diagnosis/relapse samples from 10 patients with recurrence showed that 8 of 10 paired diagnosis and relapse samples had inconsistent expression of IKAROS isoforms. The rate of IK6 expression in relapse samples from 21 relapse ALL patients was significantly higher than in the 137 children with newly diagnosed ALL (62% vs 12%, P<0.01).</p><p><b>CONCLUSIONS</b>Expression of DN IKAROS isoforms can be a poor prognostic factor in B-ALL and is closely associated with recurrence of B-ALL.</p>

Relationship between the antileukemic activity of L-asparaginase and Asn level around leukemic cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the antileukemic activity of L-asparaginase through determining the changes of 4 kinds of amino acids (Asn, Aspa, Glu and Gln) in cell culture medium.</p><p><b>METHODS</b>Following L-Asp treatment with designed concentrations and duration, the IC50 (inhibitory concentration 50%) of 8 kinds of common leukemia cell lines (U937, HL-60, Jurkat, NB4, THP-1, Namalwa, Karpass299, K562) were determined by CCK-8 assay. The changes of the 4 kinds of amino acids mentioned above were detected by high performance liquid chromatography (HPLC).</p><p><b>RESULTS</b>The asparagines in cell culture medium were rapidly exhausted when treated with 0.01 U/mL L-Asp for 4 hrs or 1 U/mL L-Asp for 5 minutes. There were significant differences in the sensitivities to L-Asp of different leukemia cell lines. The sensitivities to L-Asp of various cell lines were dose-dependent. Low concentration of L-Asp resulted in a low IC50 and the IC50 increased following the L-Asp concentration increased.</p><p><b>CONCLUSIONS</b>Different leukemia cell lines have different sensitivities to L-Asp, suggesting that exhaustion of asparagines around leukemia cells could not reflect the treatment efficacy of L-Asp. L-Asp antileukemic activity is dose-dependent, which suggests the importance of high-dose L-Asp on childhood acute lymphoblastic leukemia.</p>

The cultivation and identification of tumor stem cells from neuroblastoma derived tumor spheres / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Since the proposal of the tumor stem cell hypothesis, considerable interest has been devoted to the isolation and purification of tumor stem cells. Tumor stem cell enrichment from primary tumor derived cell spheres has been demonstrated in specific, serum-free media. This goal of this study is to establish a method of cultivating floating tumor spheres from neuroblastoma cells and to confirm that neuroblastoma spheres are rich in tumor stem cells.</p><p><b>METHODS</b>Bone marrow aspirates were obtained from pediatric patients diagnosed with stage IV neuroblastoma. Primary tumor cells were isolated and cultivated in serum-free, stem cell-selective medium. Single sphere-forming cells were cultivated under serum-free conditions; their cloning efficiency and monoclonal tumor sphere formation rates were calculated. The expression of stem cell marker genes Oct-4 and Bmi-1 was detected by RT-PCR in sphere-forming cells and parental neurolastoma cells. Sphere-forming cells were injected into the armpit of nude mice with subsequent assessment for tumor growth. Sphere-forming cells were cultivated in differentiation medium containing 5 μmol/L 13-cis retinoic acid; changes in cell morphology were observed.</p><p><b>RESULTS</b>Neuroblastoma cells formed non-adherent neurospheres under serum-free, stem cell-selective conditions after a period of 4 to 6 days. A single cell dissociated from a neurosphere could reform a monoclonal sphere; cloning efficiency and monoclonal sphere formation rates were 55.3% and 26.3%, respectively. RT-PCR results revealed heightened tumor sphere expression of Oct-4 and Bmi-1 as compared with parental tumor cells. Fourteen days after injection of 10(4) sphere-forming cells into nude mice, a neuroblastoma xenograft formed. Treatment of sphere-forming cells with 13-cis retinoic acid induced a gradual differentiation to neuronal cell morphology.</p><p><b>CONCLUSIONS</b>Neuroblastoma derived tumor spheres enrich tumor stem cells and the cultivation of primary neuroblastoma cells in serum-free, stem cell-selective medium is an effective method to dissociate and purify tumor stem cells in vitro.</p>

Relationship between asparagine synthetase expression level and cell sensitivity to L-asparaginase in human leukemic cell lines / 中国实验血液学杂志

This study was purposed to explore the relationship between asparagine synthetase (AsnS) mRNA expression level and the sensitivity of leukemic cell lines to L-asparaginase. The AsnS mRNA expression level in 8 cell lines (Jurkat, HL-60, U937, NB4, THP-1, Namalwa, Karpas299 and K562) was determined by real-time quantitative PCR (RQ-PCR) based on fluorescence dye Eva Green before and after treatment with L-Asp, and the cell proliferation rates were analyzed by CCK-8 assay. The results showed that there was a significant disparity of AsnS expression level in 8 cell lines, and there were significant increases of AsnS expression level in cells co-cultured with L-Asp (p < 0.05). Of all these eight cell lines, cells sensitive to L-asparaginase had lower AsnS expression level and cells resistant to L-asparaginase had higher AsnS expression. U937 which was the most sensitive to L-asparaginase had the lowest AsnS expression level, while K562 was natural resistant to L-asparaginase and possessed of the highest AsnS level. It is concluded that the AsnS plays a critical role in regulating cellular biological behavior after depletion of asparagine, the AsnS mRNA expression level in cells reflects the sensitivity of cells to L-Asp. The results may imply the possibility for the use of L-asparaginase in leukemia with lower AsnS expression level.

Interaction between mixed-lineage leukemia and Menin proteins / 中国实验血液学杂志

The study was aimed to investigate the subcellular localization of mixed-lineage leukemia (MLL) and Menin proteins, and to explore the interaction between these two proteins. The recombinant eukaryotic cell expression vectors of pcDNA3.1-myc-MLL and pCMV-flag-Menin were constructed respectively, and transfected into the HEK293T cells. Immunofluorescence technique was used to observe the subcellular localization of the two proteins. Co-immunoprecipitation and Western blot methods were applied to evaluate the expression and interaction of the two proteins. The results showed that MLL and Menin proteins could be co-localized in cell nuclei, and the study of binding in vivo revealed that MLL protein could be detected in the immunoprecipitation complex of anti-FLAG, while Menin proteins could also be found in the immunoprecipitation complex of anti-MYC. It is concluded that MLL and Menin proteins co-localized in cell nuclei have same location and the interaction exists between MLL and Menin proteins.

Relationship between the expression of the genes encoding the key enzymes for cytarabine metabolism and the pharmacokinetics of cytarabine in the treatment of childhood acute leukemia with high-dose cytarabine / 中华儿科杂志

<p><b>OBJECTIVE</b>It has been reported that high-dose cytarabine (HD-AraC) was very effective for childhood hematological malignancies, especially for improving the long-term survival of high-risk acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and T-cell lymphoid malignancies (T-ALL, T-cell non-Hodgkin's lymphoma). This study aimed to evaluate the pharmacokinetics of HD-AraC for childhood hematological malignancies, and the relationship between the expression of the genes coding the key enzymes for Ara-C metabolism with the outcome of the patients.</p><p><b>METHODS</b>The drug levels of Ara-C in plasma and cerebrospinal fluid were detected with HPLC while HD-AraC was used, the expression of deoxycytidine kinase (dCK) and cytidine deaminase (CDA) mRNA in human leukemia cell lines and the bone marrow cells were investigated in 48 cases of childhood hematological malignancies with RT-PCR methods, and the relationship between the expression of these enzymes mRNA and the outcome of the patients was analyzed.</p><p><b>RESULTS</b>(1) When HD-AraC was used, the plasma levels of Ara-C and Ara-U could be respectively about 50 times and 25 times higher than those obtained when the patients were treated with regular dose of Ara-C treatment, and the level of Ara-C in cerebrospinal fluid could reach about 10% of plasma level of Ara-C. (2) There were significantly different expressions of dCK mRNA in different childhood acute leukemia (AL) patients, which were markedly related to the chemotherapy results. The expression of dCK in ALL was much higher than that in AML and relapsed AL cases. There were no significant differences in expressions of dCK in T-ALL and B lineage ALL. (3) In vitro study found that the expressions of dCK and CDA mRNA did not change in leukemia cell lines incubated at different doses and times of Ara-C.</p><p><b>CONCLUSIONS</b>HD-AraC was a very effective protocol for childhood hematological malignancies for it could significantly elevate the plasma and cerebrospinal fluid drug levels. The expression of dCK may be an important factor in predicting the long-term outcomes of children with hematological malignancies. Good long-term outcomes of the childhood T-ALL could be achieved as the B lineage ALL had been treated with HD-AraC regimen. As the expression levels of dCK were much lower, it may be necessary for the treatment of AML with HD-AraC for consecutive three days.</p>

Comparative proteomic analysis of differentially expressed proteins between K562 and K562/ADM cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Multidrug resistance to chemotherapeutic agents is an important clinical problem during the treatment of leukemia. The resistance process is multifactorial. To realize the total factors involved in multidrug resistance, we analyzed the differentially expressed proteins of K562 and K562/ADM cells and we investigated one of the up-regulated proteins (CRKL) using siRNA to determine its role in K562/ADM cells.</p><p><b>METHODS</b>Altered protein expressions between K562/S (K562 ADM-sensitive cell line) and K562/ADM (K562 multidrug resistant cell line induced by adriamycin) were identified by 2D-DIGE coupled with mass spectrometry. Meanwhile, we confirmed the differential expression of CRKL and Stathmin in both K562 and K562/ADM cells by Western blot analysis. Furthermore, we used RNA interference to silence the CRKL gene expression.</p><p><b>RESULTS</b>Among the 9 differentially expressed proteins, 3 were up-regulated in K562/ADM cells, while 6 were down-regulated in the K562/ADM cells compared with its parent cell line. The expression of CRKL was up-regulated significantly in K562/ADM cells, and it can be decreased by recombinant lentivirus. Moreover, the multidrug resistance of K562/ADM cells was efficiently reversed by silence of CRKL gene expression.</p><p><b>CONCLUSIONS</b>The data provided the differentially expressed proteins in K562 and its resistant cell line and highlights the power of 2D-DIGE for the discovery of resistance markers in cancer. We found CRKL may be a new protein involved in the multidrug resistance of leukaemia cells.</p>

Study of the correlation between the expression level of asparagine synthetase and the outcome of children with acute lymphocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To determine whether the high level of asparagine synthetase (AS) expression in childhood acute lymphocytic leukemia (ALL) is associated with an inferior prognosis.</p><p><b>METHODS</b>AS mRNA level in leukemic cells from 53 newly diagnosed ALL children was measured by real time fluorescent quantitative PCR method. Patients were divided into groups according to their relapse risk and outcome, and the AS expression levels in each group were compared. The survival rates in different AS expressing level groups were estimated and compared.</p><p><b>RESULTS</b>The highest level of AS [median 17.25 (2.48-46. 82)] was observed in children failed remission, intermediate level [14.28 (3.20-54.47)] in relapsed children and the lowest level [5.08 (0.84-54.92)] in children with continuous complete remission (CCR) (P<0.05). The AS mRNA level [14.93 (2.48-54.47)] in children with poor outcome (un-remission and relapsed) was significantly higher than that in children in CCR (P<0.01). The two-year estimated disease free survival was much lower in children with high AS expression (53.8%) than in those with low AS expression (84.6%) (P<0.05).</p><p><b>CONCLUSION</b>High expression of AS is associated with a poor outcome in ALL children.</p>

Single nucleotide polymorphism in the promoter region of asparagine synthetase and its impact on the gene expression / 中国实验血液学杂志

High expression of cellular asparagine synthetase (AS) is a causative factor for the resistance of leukemic cell to L-asparaginase therapy. This study was aimed to find single nucleotide polymorphism (SNPs) in the promotor region of asparagine synthetase (AS) gene and to determine if these SNPs have influence on the transcriptional activity of AS promotor. The DNA sequences of AS promoter (pAS) from 82 leukemic children and 45 controls were determined to screen for SNPs in this region and the AS mRNA level in these samples was quantified using real-time PCR assay. The results indicated that three SNPs were found in the sequenced pAS fragment. They were -239C/T, -92G/A and -62A/T respectively. The frequency of -92A allele was higher in leukemic samples than that in nonleukemic control (P<0.05). The gene expression level differed among the individuals with genotype of the -92G/A SNP, and the descending order was as follows: GA heterozygote > AA homozygote > GG homozygote. It is concluded that some features in leukemia might associate with SNP on -92A locus, and this SNP in pAS can be one of the factors influencing transcriptional activity of AS gene. The existence of the -92A allele variant contributes to a high expression of AS gene.

Effect of vascular endothelial growth factor on apoptosis and expression of Bcl-2 and Mcl-1 in acute leukemia cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effect of vascular endothelial growth factor (VEGF) on the apoptosis of human acute leukemia HL-60 cell line and to analyze the role of the related apoptosis genes, such as Bcl-2 and Mcl-1, in the process of apoptosis of human acute leukemia cells.</p><p><b>METHODS</b>HL-60 cells were treated with different concentrations of VEGF (2 microg/L, 20 microg/L or 100 microg/L ) or 20 mg/L of etoposide (VP16, an apoptosis inducter) alone or VEGF plus VP16. After 18 hrs of treatment, the apoptosis rate of HL-60 cells was detected by single-cell gel electrophoresis and flow cytometry. The expressions of Bcl-2 and Mcl-1 of HL-60 cells were detected by RT-PCR. The Control group did not receive any treatment. Immunocytochemistry was used to detect the VEGF and Mcl-1 protein in bone marrow cells from 8 patients with newly diagnosed or relapsed leukemia, 14 leukemia patients in complete remission, and from 5 normal children.</p><p><b>RESULTS</b>Different concentrations of VEGF markedly inhibited the apoptosis of HL-60 cells and decreased the apoptosis induced by VP16 exposure. The Bcl-2 and Mcl-1 mRNA and protein in HL-60 cells treated with VEGF were significantly higher than those in the Control group. The expressions of VEGF and Mcl-1 protein in bone marrow cells of the newly diagnosed and relapsed patients were significantly higher than in patients in complete remission.</p><p><b>CONCLUSION</b>VEGF can inhibit the apoptosis of HL-60 cells possibly through increasing the expressions of Bcl-2 and Mcl-1 mRNA and protein, which may represent one of the mechanisms responsible for human acute leukemia. The expressions of VEGF, Bcl-2 and Mcl-1 might be used as the markers for the prognostic evaluation of leukemia.</p>