RESUMEN
BACKGROUND: HSCT is the only proven curative therapy for JMML. Matching donor and recipient HLA alleles is considered optimal to reduce the risk of GVHD after HSCT but is not always possible. Only a limited number of studies have compared the influence of HLA disparities on HSCT outcomes for patients with JMML. METHODS: We conducted a retrospective study among 47 children with JMML who received related or unrelated unmanipulated HSCT (March 2010-October 2018). Among our participants, 27 (57.4%) donor-recipient pairs had 0-1 HLA disparities (Group 1: HLA-matched or ≤1 allele/antigen mismatch donor) and 20 (42.6%) had ≥2 HLA disparities (Group 2: 2-3 mismatched/haploidentical donors). RESULTS: The median follow-up period was 26.0 months (range: 1-105 months), and the 5-year probabilities of DFS and RI for the whole cohort were 54.6 ± 7.7% and 34.8 ± 15.0%, respectively. Compared to Group 1, Group 2 patients had a significantly lower RI (5.3 ± 10.5% vs 55.5 ± 20.9%, P Ë .001), though similar rates of grade II-IV acute GVHD (60.0 ± 22.4% vs 33.3 ± 18.2%, P = .08), grade III-IV acute GVHD (25.0 ± 19.5% vs 7.4 ± 10.1%, P = .08), chronic GVHD (30.0 ± 20.9% vs 34.9 ± 18.8%, P = .85), NRM (20.0 ± 18.0% vs 3.9 ± 7.7%, P = .07), and DFS (74.4 ± 9.9% vs 41.3 ± 10.0%, P = .08). CONCLUSIONS: Disease relapse remains the major cause of treatment failure in JMML patients, especially in patients receiving HLA-matched and limited HLA-mismatched HSCT. Our findings suggest that donor-recipient HLA disparities may improve the outcome of HSCT in children with JMML.
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Selección de Donante , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Prueba de Histocompatibilidad , Leucemia Mielomonocítica Juvenil/terapia , Biomarcadores , Niño , Preescolar , China , Femenino , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Lactante , Estimación de Kaplan-Meier , Leucemia Mielomonocítica Juvenil/inmunología , Leucemia Mielomonocítica Juvenil/mortalidad , Masculino , Modelos de Riesgos Proporcionales , Recurrencia , Estudios Retrospectivos , Prevención Secundaria , Donantes de Tejidos , Resultado del TratamientoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.
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Regulación Leucémica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Transcriptoma , Adulto , Niño , Estudios de Cohortes , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Células HEK293 , Humanos , Células Jurkat , Estimación de Kaplan-Meier , MutaciónRESUMEN
OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: Expression of DN IKAROS isoforms can be a poor prognostic factor in B-ALL and is closely associated with recurrence of B-ALL.
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Factor de Transcripción Ikaros/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Pronóstico , Isoformas de Proteínas/genéticaRESUMEN
OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.
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Factores de Transcripción de Tipo Kruppel/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Adolescente , Línea Celular Tumoral , Niño , Preescolar , Femenino , Humanos , Lactante , Factores de Transcripción de Tipo Kruppel/análisis , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Pronóstico , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mismatch repair (MMR) deficiency has been linked to thiopurine resistance and hypermutation in relapsed acute lymphoblastic leukemia (ALL). However, the repair mechanism of thiopurine-induced DNA damage in the absence of MMR remains unclear. Here, we provide evidence that DNA polymerase ß (POLB) of base excision repair (BER) pathway plays a critical role in the survival and thiopurine resistance of MMR-deficient ALL cells. In these aggressive resistant ALL cells, POLB depletion and its inhibitor oleanolic acid (OA) treatment result in synthetic lethality with MMR deficiency through increased cellular apurinic/apyrimidinic (AP) sites, DNA strand breaks and apoptosis. POLB depletion increases thiopurine sensitivities of resistant cells, and OA synergizes with thiopurine to kill these cells in ALL cell lines, patient-derived xenograft (PDX) cells and xenograft mouse models. Our findings suggest BER and POLB's roles in the process of repairing thiopurine-induced DNA damage in MMR-deficient ALL cells, and implicate their potentials as therapeutic targets against aggressive ALL progression.
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ADN Polimerasa beta , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Humanos , Ratones , Daño del ADN , ADN Polimerasa beta/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Mutaciones Letales Sintéticas , Reparación de la Incompatibilidad de ADN/genéticaRESUMEN
OBJECTIVE: To reduce the risk of therapy related complication during the treatment and keeps the long term event free survival, and to evaluate the results and risk factors of SCMC-lymphoblastic leukemia (ALL)-2005 protocol. METHODS: Designed the new protocol SCMC-ALL-2005 based on the previous protocol XH-99 for ALL. Divided the patients into low, median and high risk groups depends on risk factors including day 33 and 55 minimal residual disease (MRD) level. The higher risk group, the more intensive therapy was given. All the cases were registed on pediatric oncology network database (POND). All the abandonment patients were counted as event. From May 1(st) 2005 to April 30(th) 2009, 351 children who were newly diagnosed as B lineage ALL were enrolled in this study. The prognoses relating to risk grouping, age, mutation gene and MRD level were analyzed. RESULTS: Up to June 30, 2011, 273 patients were followed up with median time 49 months (range 26 to 74 months). Three hundred and forty-five patients (98.29%) achieved complete remission on day 35 induction. 12 cases were younger than 1 year old (3.42%), 285 cases between 1 and 9 years old (81.20%), 54 cases 10 to 18 years old (15.38%). Five year event-free survival (EFS) was 34%, 72% and 63%, respectively. One hundred and fifty-six cases belonged to lowered risk (44.44%), 177 to middle risk (50.43%) and 18 to higher risk (5.13%). Five year EFS was 78%, 64% and 30%, respectively. In this study, 18 patients were detected positive for BCR/ABL, 3 for MLL/AF4, 16 for PBX/E2A, and 36 for TEL/AML. The 5 year EFS were 11%, 66%, 75% and 74%, respectively. A total of 300 cases were tested for MRD levels on day 35. Of them, 241 cases were with MRD ≤ 0.01% (negative), and 59 cases > 0.01% (positive). The 5 year relapse free survival (RFS) was 79% and 58%, respectively. Total 6 patients died of complication (1.71%). 18 patients were abundant treatment with no disease progress. 70 patients relapsed (19.94%), including 52 bone marrow, 8 central nerve system (CNS), 1 both in bone marrow and CNS, 1 second cancer (M(4)) and 8 testis. Five year overall survival (OS) and EFS are 84% and 69%. CONCLUSIONS: The risk of therapy related death is low with the protocol SCMC-ALL-2005. MRD affects the prognosis. The long term prognosis is poor for high risk group, with BCR/ABL and positive MRD.
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Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma de Burkitt/terapia , Neoplasia Residual/terapia , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios ProspectivosRESUMEN
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.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores Quiméricos de Antígenos , Adolescente , Antígenos CD19 , Niño , Preescolar , Enfermedad Crónica , Ferritinas , Humanos , Inmunoterapia Adoptiva , Metilprednisolona , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos/metabolismo , Recurrencia , Linfocitos TRESUMEN
BACKGROUND AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Diferenciación Celular/efectos de los fármacos , Células Madre Neoplásicas/patología , Neuroblastoma/patología , Esferoides Celulares/patología , Animales , Técnicas de Cultivo de Célula/métodos , Niño , Medio de Cultivo Libre de Suero , Humanos , Isotretinoína/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
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Asparaginasa/farmacología , Asparagina/análisis , Leucemia/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Humanos , Leucemia/metabolismo , Leucemia/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), resulting in a PML-RARA fusion that is the master driver of APL. A few cases that cannot be identified with PML-RARA by using conventional methods (karyotype analysis, FISH, and RT-PCR) involve abnormal promyelocytes that are fully in accordance with APL in morphology, cytochemistry, and immunophenotype. To explore the mechanisms involved in pathogenesis and recurrence of morphologically diagnosed APL, we performed comprehensive variant analysis by next-generation sequencing in 111 pediatric patients morphologically diagnosed as APL. Structural variant (SV) analysis in 120 DNA samples from both diagnosis and relapse stage identified 95 samples with RARA rearrangement (including 94 with PML-RARA and one with NPM-RARA) and two samples with KMT2A rearrangement. In the eligible 13 RNA samples without any RARA rearrangement at diagnosis, one case each with CPSF6-RARG, NPM1-CCDC28A, and TBC1D15-RAB21 and two cases with a TBL1XR1-RARB fusion were discovered. These uncovered fusion genes strongly suggested their contributions to leukemogenesis as driver alternations and APL phenotype may arise by abnormalities of other members of the nuclear receptor superfamily involved in retinoid signaling (RARB or RARG) or even by mechanisms distinct from the formation of aberrant retinoid receptors. Single-nucleotide variant (SNV) analysis in 77 children (80 samples) with RARA rearrangement showed recurrent alternations of primary APL in FLT3, WT1, USP9X, NRAS, and ARID1A, with a strong potential for involvement in pathogenesis, and WT1 as the only recurrently mutated gene in relapsed APL. WT1, NPM1, NRAS, FLT3, and NSD1 were identified as recurrently mutated in 17 primary samples without RARA rearrangement and WT1, NPM1, TP53, and RARA as recurrently mutated in 9 relapsed samples. The survival of APL with RARA rearrangement is much better than without RARA rearrangement. Thus, patients morphologically diagnosed as APL that cannot be identified as having a RARA rearrangement are more reasonably classified as a subclass of AML other than APL, and individualized treatment should be considered according to the genetic abnormalities.
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Biomarcadores de Tumor/genética , Células Precursoras de Granulocitos/patología , Leucemia Promielocítica Aguda/genética , Mutación , Recurrencia Local de Neoplasia/genética , Proteínas de Fusión Oncogénica/genética , Translocación Genética , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células Precursoras de Granulocitos/metabolismo , Humanos , Lactante , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Masculino , Recurrencia Local de Neoplasia/diagnóstico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Nucleofosmina , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Bone marrow (BM) niche responds to chemotherapy-induced cytokines secreted from acute lymphoblastic leukemia (ALL) cells and protects the residual cells from chemotherapeutics in vivo. However, the underlying molecular mechanisms for the induction of cytokines by chemotherapy remain unknown. Here, we found that chemotherapeutic drugs (e.g., Ara-C, DNR, 6-MP) induced the expression of niche-protecting cytokines (GDF15, CCL3 and CCL4) in both ALL cell lines and primary cells in vitro. The ATM and NF-κB pathways were activated after chemotherapy treatment, and the pharmacological or genetic inhibition of these pathways significantly reversed the cytokine upregulation. Besides, chemotherapy-induced NF-κB activation was dependent on ATM-TRAF6 signaling, and NF-κB transcription factor p65 directly regulated the cytokines expression. Furthermore, we found that both pharmacological and genetic perturbation of ATM and p65 significantly decreased the residual ALL cells after Ara-C treatment in ALL xenograft mouse models. Together, these results demonstrated that ATM-dependent NF-κB activation mediated the cytokines induction by chemotherapy and ALL resistance to chemotherapeutics. Inhibition of ATM-dependent NF-κB pathway can sensitize ALL to chemotherapeutics, providing a new strategy to eradicate residual chemo-resistant ALL cells.
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Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos , Línea Celular Tumoral , Niño , Citocinas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Doxorrubicina/farmacología , Células K562/química , Proteínas de Neoplasias/análisis , Proteómica , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Resistencia a Múltiples Medicamentos , Humanos , Células K562/efectos de los fármacos , Datos de Secuencia Molecular , Proteínas Nucleares/análisis , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Estatmina/análisisRESUMEN
OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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Apoptosis/efectos de los fármacos , Leucemia/patología , Proteínas de Neoplasias/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Factor A de Crecimiento Endotelial Vascular/farmacología , Citometría de Flujo , Células HL-60 , Humanos , Inmunohistoquímica , Leucemia/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.
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Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Genómica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcriptoma , Adolescente , Adulto , Anciano , Médula Ósea/patología , Niño , Preescolar , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Persona de Mediana Edad , Mutación , Tasa de Mutación , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Adulto JovenRESUMEN
B cell acute lymphoblastic leukemia (B-ALL) is the most common hematological malignancy diagnosed in children, and blockade of the abnormally activated PI3Kδ displayed promising outcomes in B cell acute or chronic leukemias, but the mechanisms are not well understood. Here we report a novel PI3Kδ selective inhibitor X-370, which displays distinct binding mode with p110δ and blocks constitutively active or stimulus-induced PI3Kδ signaling. X-370 significantly inhibited survival of human B cell leukemia cells in vitro, with associated induction of G1 phase arrest and apoptosis. X-370 abrogated both Akt and Erk1/2 signaling via blockade of PDK1 binding to and/or phosphorylation of MEK1/2. Forced expression of a constitutively active MEK1 attenuated the antiproliferative activity of X-370. X-370 preferentially inhibited the survival of primary pediatric B-ALL cells displaying PI3Kδ-dependent Erk1/2 phosphorylation, while combined inhibition of PI3Kδ and MEK1/2 displayed enhanced activity. We conclude that PI3Kδ inhibition led to abrogation of both Akt and Erk1/2 signaling via a novel PI3K-PDK1/MEK1/2-Erk1/2 signaling cascade, which contributed to its efficacy against B-ALL. These findings support the rationale for clinical testing of PI3Kδ inhibitors in pediatric B-ALL and provide insights needed to optimize the therapeutic strategy.
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Bencimidazoles/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Purinas/farmacología , Apoptosis/efectos de los fármacos , Bencimidazoles/química , Western Blotting , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Humanos , Inmunoprecipitación , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/química , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Chromosome rearrangements and fusion genes present major portion of leukemogenesis and contribute to leukemic subtypes. It is practical and helpful to detect the fusion genes in clinic diagnosis of leukemia. Present application of reverse transcription polymerase chain reaction (RT-PCR) method to detect the fusion gene transcripts is effective, but time- and labor-consuming. To set up a simple and rapid system, we established a method that combined multiplex RT-PCR and microarray. We selected 15 clinically most frequently observed chromosomal rearrangements generating more than 50 fusion gene variants. Chimeric reverse primers and chimeric PCR primers containing both gene-specific and universal sequences were applied in the procedure of multiplex RT-PCR, and then the PCR products hybridized with a designed microarray. With this approach, among 200 clinic samples, 63 samples were detected to have gene rearrangements. All the detected fusion genes positive and negative were validated with RT-PCR and Sanger sequencing. Our data suggested that the RT-PCR-microarray pipeline could screen 15 partner gene pairs simultaneously at the same accuracy of the fusion gene detection with regular RT-PCR. The pipeline showed effectiveness in multiple fusion genes screening in clinic samples.
Asunto(s)
Cromosomas Humanos/genética , Pruebas Genéticas , Leucemia/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Translocación Genética , Células HL-60 , Humanos , Leucemia/diagnóstico , Proteínas de Fusión Oncogénica/genética , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To summarize long-term outcomes of childhood lymphoblastic lymphoma (LBL) with protocol CCCG-97 and -2002. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Pronóstico , Estudios Prospectivos , Resultado del TratamientoRESUMEN
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.
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Asparaginasa/farmacología , Aspartatoamoníaco Ligasa/metabolismo , Leucemia/enzimología , Asparaginasa/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
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.
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Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/genética , Mapeo Cromosómico , Proteínas de Unión al ADN , Vectores Genéticos , Células HEK293 , Proteínas de Homeodominio , Humanos , Mapeo de Interacción de Proteínas , TransfecciónRESUMEN
OBJECTIVE: To determine whether the high level of asparagine synthetase (AS) expression in childhood acute lymphocytic leukemia (ALL) is associated with an inferior prognosis. METHODS: 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. RESULTS: 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). CONCLUSION: High expression of AS is associated with a poor outcome in ALL children.