Clinical, biological, and outcome features of P2RY8-CRLF2 and CRLF2 over-expression in pediatric B-cell precursor acute lymphoblastic leukemia according to the CCLG-ALL 2008 and 2018 protocol.

OBJECTIVES: CRLF2 alterations are associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This study aimed to explore the clinical, biological, and outcome features of pediatric BCP-ALL with CRLF2 abnormalities. METHODS: This study enrolled 630 childhood BCP-ALLs treated on CCLG-ALL 2008 or 2018 protocol. P2RY8-CRLF2 was determined by Sanger sequencing and CRLF2 expression was evaluated by qRT-PCR. The correlation between clinical, biological features and outcomes with P2RY8-CRLF2 or CRLF2 over-expression were analyzed. RESULTS: P2RY8-CRLF2 and CRLF2 over-expression were found in 3.33% and 5.71% respectively. P2RY8-CRLF2 was associated with male, higher frequency of CD7 expression, high WBC and MRD before consolidation. CRLF2 over-expression showed ETV6-RUNX1- , higher frequency of CD22, CD34, CD66c, CD86 expression, hyperdiploidy and high MRD at early treatment. The lower overall survival (OS) was found in patients with P2RY8-CRLF2 and confined only in IR group. Furthermore, adverse event-free survival and OS of P2RY8-CRLF2 were discovered comparing to those without known fusions or treated on CCLG-ALL 2008 protocol. However, P2RY8-CRLF2 was not confirmed as independent prognostic factors and no prognostic impact of CRLF2 over-expression was found. CONCLUSIONS: These findings indicate P2RY8-CRLF2 identifies a subset of patients with specific features and adverse outcomes that could be improved by risk-directed treatment.

ß-catenin promotes MTX resistance of leukemia cells by down-regulating FPGS expression via NF-κB.

BACKGROUND: Aberrant activation of ß-catenin has been shown to play important roles in the chemoresistance of acute lymphoblastic leukemia (ALL), but the involvement and mechanism of ß-catenin in methotrexate (MTX) resistance is poorly understood. In the present study, we demonstrate a critical role of ß-catenin-NF-κB-FPGS pathway in MTX resistance in the human T-lineage ALL cell lines. METHODS: Lentivirus sh-ß-catenin was used to silence the expression of ß-catenin. Flow cytometry was performed to detect apoptosis after MTX treatment. Western blot, real-time PCR, Co-immunoprecipitation (Co-IP), Chromatin immunoprecipitation (ChIP), Re-ChIP, and Luciferase assay were utilized to investigate the relationship among ß-catenin, nuclear factor (NF)-κB, and folypoly-γ-glutamate synthetase (FPGS). RESULTS: Depletion of ß-catenin significantly increased the cytotoxicity of MTX. At the molecular level, knockdown of ß-catenin caused the increase of the protein level of FPGS and NF-κB p65. Furthermore, ß-catenin complexed with NF-κB p65 and directly bound to the FPGS promoter to regulate its expression. In addition, ß-catenin repression prolonged the protein turnover of FPGS. CONCLUSIONS: Taken together, our results demonstrate that ß-catenin may contribute to MTX resistance in leukemia cells via the ß-catenin-NF-κB-FPGS pathway, posing ß-catenin as a potential target for combination treatments during ALL therapy.

Clinical-biological characteristics and treatment outcomes of pediatric pro-B ALL patients enrolled in BCH-2003 and CCLG-2008 protocol: a study of 121 Chinese children.

BACKGROUND: Although leukemic blast cells of Pro-B cell acute lymphoblastic leukemia (ALL) are arrested at the same stage of B cell differentiation, the immature B cell subtype is still biologically heterogeneous and is associated with diverse outcomes. This study aimed to explore the clinical-biological characteristics of pediatric pro-B ALL and factors associated with outcomes. METHODS: This study enrolled 121 pediatric patients aged 6 months to 14 years with newly diagnosed CD19+CD10- pro-B cell acute lymphoblastic leukemia (pro-B ALL) treated at Beijing Children's Hospital from March 2003 to October 2018. Genetic abnormalities, immunophenotypic markers, minimal residual disease (MRD) at early treatment stage and long-term outcomes of children treated on two consecutive protocols were analyzed. RESULTS: KMT2A rearrangements were the most frequent abnormalities (incidence rate 33.06%), and were associated with lower frequency of CD13, CD33, CD22 and CD34 expression and higher frequency of CD7 and NG2 expression. Higher frequency of CD15 and CD133 expression was found in KMT2A-AFF1 + patients, exclusively. Presence of CD15 and absence of CD34 at diagnosis correlated with the high burden of MRD at the early stage of treatment. Outcomes were more favorable in patients older than 1 year, with absence of CD20 expression and KMT2A rearrangements, and with MRD lower than 1% at the end of induction and 0.1% before consolidation. Increased intensity of chemotherapy based on MRD analysis did not improve outcomes significantly (5-year EFS 73.9 ± 6.5% for BCH-2003 and 76.1 ± 5.3% for CCLG-2008, P = 0.975). Independent adverse prognostic factors were MRD ≥ 0.1% before consolidation and presence of KMT2A gene rearrangements (odds ratios [ORs] 9.424 [95% confidence interval (CI) 3.210, 27.662; P < 0.001]; 4.142 [1.535, 11.715, P = 0.005]; respectively). CONCLUSIONS: Pediatric pro-B ALL is a heterogeneous disease. Genetic analysis and MRD evaluation can predict patients with dismal prognosis; however, intensive chemotherapy alone does not improve outcomes of these patients and targeted therapy or hematopoietic stem cell transplantation may be required.

The prognostic potential of coilin in association with p27 expression in pediatric acute lymphoblastic leukemia for disease relapse.

BACKGROUND: Cajal body (CB) is a nucleic organelle where small nuclear ribonucleoproteins undergo modification, maturation, splicing and/or assembly. Coilin is the marker structural protein of CBs. The expression level and cellular localization of coilin is sensitive to chemotherapeutic reagents, such as cisplatin. The gene of cyclin-dependent kinase inhibitor 1B (p27) is located with a high incidence translocation region of leukemic chromosomes, and its expression was of prognosis values in a variety of adult leukemia types. The exact profile and associated functions of coilin, as well as p27, in children's acute lymphoblastic leukemia (ALL) is obscure. METHODS: Bone marrow samples from 144 patients with ALL were collected. The expression levels of coilin and p27 were detected by qRT-PCR. The patient cohort was divided into low and high groups of coilin and p27 respectively. The prognosis and clinicobiological characteristics of different groups were investigated, especially focused on the treatment outcome. Leukemia cells of Reh or RS4;11 were exposed to different concentrations of DNR, prior to the detection for morphological changes of coilin by immunofluorescence. In Reh cells, lentivirus sh-coilin was used to silence coilin expression. Western blotting was used to detect coilin and p27 expression; flow cytometry was used for cell cycle and apoptosis assay; MTS method was used for measuring cell viability to examine the drug sensitivity of DNR. RESULTS: In this study, we found that daunorubicin was able to induce significant morphological changes of CBs in Reh and RS4;11 cells. Knockdown the expression of coilin increased the sensitivity to daunorubicin and inhibited the expression of p27 in Reh cells, and led to increased apoptosis. Importantly, not only the levels of coilin and p27 mRNA expression at initial diagnosis ALL children are markedly higher than those at complete remission (CR), but also both coilin and p27 expression in the relapsed patients was observed significantly higher comparing to the continuous CR patients. The 4-year EFS and RFS indicated that low levels of both coilin and p27 group favored better prognosis (p < 0.05). CONCLUSIONS: Our results indicated that consideration of coilin and p27 levels could be a prognostic reference for predicting the outcome of pediatric ALL patients, especially for disease recurrence. Reduction of coilin expression was sufficient to increase the sensitivity of leukemic cells to daunorubicin treatments, and during which possibly involved functions of p27 in cell cycle regulation and its effects on cell apoptosis.

MYCN amplification predicts poor prognosis based on interphase fluorescence in situ hybridization analysis of bone marrow cells in bone marrow metastases of neuroblastoma.

BACKGROUND: MYCN gene amplification is related to risk stratification. Therefore it is important to identify accurately the level of the MYCN gene as early as possible in neuroblastoma (NB); however, for patients with bone marrow (BM) metastasis who need chemotherapy before surgery, timely detection of the MYCN gene is not possible due to the unavailability of primary tumors. METHODS: MYCN gene status was evaluated in 81 BM metastases of NB by interphase fluorescence in situ hybridization (FISH) analysis of BM cells. The clinicobiological characteristics and prognostic impact of MYCN amplification in NB metastatic to BM were analyzed. RESULTS: MYCN amplification was found in 16% of patients with metastases, and the results were consistent with the primary tumors detected by pathological tissue FISH. MYCN amplification was associated with age, lactate dehydrogenase (LDH) levels and prognosis (P = 0.038, P < 0.001, P = 0.026). Clinical outcome was poorer in patients with MYCN amplification than in those without amplification (3-year EFS 28.8 ± 13.1 vs. 69.7 ± 5.7%, P = 0.005; 3-year OS 41.5 ± 14.7 vs. 76.7 ± 5.5%, P = 0.005). CONCLUSIONS: MYCN amplification predicts a poor outcome in NB metastatic to BM, and interphase FISH of bone marrow cells provides a timely direct and valid method to evaluate the MYCN gene status.

Low CREBBP expression is associated with adverse long-term outcomes in paediatric acute lymphoblastic leukaemia.

OBJECTIVES: CREBBP alterations are associated with many diseases including leukaemia. However, CREBBP expression and its clinical relevance in paediatric acute lymphoblastic leukaemia have not been elucidated. METHODS: We studied CREBBP mRNA expression in 349 patients treated with either the BCH-2003 or CCLG-2008 protocol. Using a receiver operating characteristic curve, patients were divided into low- or high-CREBBP. The association among clinicobiological characteristics, outcomes and CREBBP level was analysed. RESULTS: Low expression of CREBBP (<1.0) at diagnosis was found in 97.7% of patients and increased significantly after complete remission. Low-CREBBP patients were associated with unfavourable clinical presentations, poor prednisone response and high minimal residual disease (>10-2 ) after induction. We found significantly poorer event-free survival (EFS) and overall survival (OS) in low-CREBBP group whether administered BCH-2003 or CCLG-2008. Low-CREBBP was an inferior independent prognostic factor in BCH-2003; patients with low-CREBBP had better outcomes on an intermediate-risk regimen than a standard-risk regimen involving the CCLG-2008 protocol. Patients stratified to high-risk with low-CREBBP had the worst EFS and OS. CONCLUSIONS: These findings indicate that low-CREBBP is predictive of unfavourable outcomes; thus, a more intensive treatment protocol is necessitated for standard-risk patients with insufficient CREBBP and that a specific target therapy is necessitated for high-risk patients.

FPGS rs1544105 polymorphism is associated with treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Folypolyglutamate synthase (FPGS) catalyzes the polyglutamation of folates and antifolates, such as methotrexate (MTX), to produce highly active metabolites. FPGS tag SNP rs1544105C > T is located in the gene promoter. The aim of the present study was to investigate the impact of rs1544105 polymorphism on the treatment outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: This study enrolled 164 children with BCP-ALL. We genotyped the FPGS SNP rs1544105, and analyzed the associations between its genotypes and treatment outcome. We also examined FPGS mRNA levels by real-time PCR in 64 of the 164 children, and investigated the function of this polymorphism on gene expression. RESULTS: We found significantly poor relapse-free survival (RFS) (p = 0.010) and poor event-free survival (EFS) (p = 0.046) in carriers of CC genotype. Multivariable Cox regression analyses adjusted for possible confounding variables showed that, relative to the CT + TT genotypes, the CC genotype was an independent prognostic factor for poor RFS (hazard ratio [HR], 4.992.; 95% CI, 1.550-16.078; p = 0.007). No association was found between any toxicity and rs1544105 polymorphism. Quantitative PCR results showed that individuals with the T allele had lower levels of FPGS transcripts. CONCLUSIONS: Our study indicates that FPGS rs1544105C > T polymorphism might influence FPGS expression and affect treatment outcome in BCP-ALL patients.

Clinical features, early treatment responses, and outcomes of pediatric acute lymphoblastic leukemia in China with or without specific fusion transcripts: a single institutional study of 1,004 patients.

Acute lymphoblastic leukemia (ALL) with distinct fusion transcripts has unique clinical features. In this study, the incidence, clinical characteristics, early treatment response, and outcomes of 1,004 Chinese pediatric ALLs were analyzed. Patients with TEL-AML1 and E2A-PBX1 fusion genes or other B cell precursor ALLs (BCP-ALL) had favorable clinical features, were sensitive to prednisone, had low minimal residual disease (MRD), and an excellent prognosis, with a 5-year event-free survival (EFS) of 84-92%. T-ALL was associated with a high WBC, increased age, more central nervous system involvement, a poor prednisone response, and high MRD, with a 5-year EFS of 68.4 ± 5.2%. Patients with BCR-ABL and MLL rearrangements usually had adverse clinical presentations and treatment responses, and a dismal prognosis, with 5-year EFS of 27.3 and 57.4%, respectively. We also showed that BCR-ABL and MLL rearrangements, the prednisone response, and MRD were independent prognostic factors. Interestingly, the BCH-2003 protocol resulted in a better outcome for E2A-PBX1(+) patients than the CCLG-2008 protocol. Intermediate and late relapses were more common in TEL-AML1(+) patients and other BCP-ALLs compared with other subgroups (P = 0.018). Therefore, this study suggests that a fusion gene-specific chemotherapy regimen and/or targeted therapy should be developed to improve further the cure rate of pediatric ALL.

MYCN amplification plus 1p36 loss of heterozygosity predicts ultra high risk in bone marrow metastatic neuroblastoma.

BACKGROUND: This study aimed to better understand the prognostic effect of multiple genetic markers and identify more subpopulations at ultra high risk of poor outcome in bone marrow (BM) metastatic neuroblastoma (NB). METHODS: We screened the MYCN, 1p36 and 11q23 loss of heterozygosity (LOH) statuses of 154 patients by interphase fluorescence in situ hybridization of BM cells. The clinical characteristics of patients with the three markers and their associations with prognosis were analysed. RESULTS: MYCN amplification and LOH at 1p36 and 11q23 were identified in 16.2%, 33.1% and 30.5% of patients, respectively. There were strong associations between MYCN amplification and 1p36 LOH as well as 11q23 LOH. Both MYCN amplification and 1p36 LOH were strongly associated with high levels of lactate dehydrogenase (LDH) and neuron-specific enolase, more than 3 metastatic organs, and more events. 11q23 LOH occurred mainly in patients older than 18 months, and those who had high LDH levels. In univariate analysis, patients with MYCN amplification had poorer prognosis than those without. Patients with 1p36 LOH had a 3-year event-free survival (EFS) and overall survival lower than those without. 11q23 LOH was associated with poorer EFS only for patients without MYCN amplification. In a multivariate model, MYCN amplification was independently associated with decreased EFS in all cohorts. 11q23 LOH was an independent prognostic factor for patients without MYCN amplification, whereas 1p36 LOH was not an independent marker regardless of MYCN amplification. Compared with all cohorts, patients with both MYCN amplification and 1p36 LOH had the worst outcome and clinical features. CONCLUSIONS: Patients with both MYCN amplification and 1p36LOH had the worst survival rate, indicating an ultra high-risk group. Our results may be applied in clinical practice for accurate risk stratification in future studies.

Chromosome 10 abnormality predicts prognosis of neuroblastoma patients with bone marrow metastasis.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor in children. It is known for high heterogeneity and concealed onset. In recent years, the mechanism of its occurrence and development has been gradually revealed. The purpose of this study is to summarize the clinical characteristics of children with NB and abnormal chromosome 10, and to investigate the relationship between the number and structure of chromosome 10 abnormalities and NB prognosis. METHODS: Chromosome G-banding was used at the time of diagnosis to evaluate the genetics of chromosomes in patients with NB and track their clinical characteristics and prognosis. All participants were diagnosed with NB in the Medical Oncology Department of the Beijing Children's Hospital from May 2015 to December 2018 and were followed up with for at least 1 year. RESULTS: Of all 150 patients with bone marrow metastases, 42 were clearly diagnosed with chromosomal abnormalities. Thirteen patients showed abnormalities in chromosome 10, and chromosome 10 was the most commonly missing chromosome. These 13 patients had higher LDH and lower OS and EFS than children with chromosomal abnormalities who did not have an abnormality in chromosome 10. Eight patients had both MYCN amplification and 1p36 deletion. Two patients had optic nerve damage and no vision, and one patient had left supraorbital metastases 5 months after treatment. CONCLUSIONS: The results indicated that chromosome 10 might be a new prognostic marker for NB. MYCN amplification and 1p36 deletion may be related to chromosome 10 abnormalities in NB. Additionally, NB patients with abnormal chromosome 10 were prone to orbital metastases.

Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells.

Low expression levels of CREB­binding protein (CREBBP) have been demonstrated to be associated with high minimal residual disease at the end of induction therapy and adverse long­term outcomes in pediatric patients with acute lymphoblastic leukemia (ALL). However, the effect of low CREBBP expression on the prognosis of ALL has not yet been investigated. In the present study, CREBBP was downregulated and overexpressed in ALL cell lines (Jurkat and Reh). Sensitivity to chemotherapy and cell proliferation activity was determined via a Cell Counting Kit­8 assay. Cell cycle analysis was performed using flow cytometry. Immunofluorescence confocal microscopy and co­immunoprecipitation (Co­IP) assays were performed to determine the interaction between CREBBP and E2F transcription factor 3a (E2F3a). The binding of CREBBP to downstream gene caspase 8 associated protein 2 (CASP8AP2) promoters was assessed using a chromatin immunoprecipitation assay, and mRNA expression levels were detected via reverse transcription­quantitative PCR. Western blot analysis was performed to detect protein expression of CREBBP, E2F3a and CASP8AP2. Downregulation of CREBBP increased the IC50 value of daunorubicin; however, no significant affects were observed on the IC50 values of vincristine and L­asparaginase. Furthermore, downregulation of CREBBP notably inhibited leukemia cell proliferation, accumulated cells in the G0/G1 phase and decreased cell proportions in the S and G2/M phases. Co­IP analysis demonstrated that CREBBP interacted with E2F3a, a transcription factor involved in G1/S transition. Immunofluorescence confocal microscopy indicated co­localization of CREBBP and E2F3a at the cell nucleus. Furthermore, E2F3a protein expression decreased in CREBBP RNA interference treated Jurkat and Reh cells. CASP8AP2, a target gene of E2F3a, was also identified to be a downstream gene of CREBBP. In addition, decreased IC50 value and cell proportions in the G0/G1 phase, accelerated cell proliferation and upregulated E2F3a and CASP8AP2 expression were exhibited in CREBBP overexpressed cells. Taken together, the results of the present study suggested that CREBBP downregulation affects proliferation and cell cycle progression in leukemia cells, potentially via the interaction and regulation of E2F3a, resulting in chemotherapy resistance. Thus, targeting CREBBP may be a therapeutic strategy for treating pediatric patients with ALL.

Chromosome band 11q23 deletion predicts poor prognosis in bone marrow metastatic neuroblastoma patients without MYCN amplification.

BACKGROUND: Interphase fluorescence in situ hybridization (FISH) of bone marrow cells has been confirmed to be a direct and valid method to assess the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification in patients with bone marrow metastatic neuroblastoma. MYCN amplification alone, however, is insufficient for pretreatment risk stratification. Chromosome band 11q23 deletion has recently been included in the risk stratification of neuroblastoma. In the present study, we aimed to evaluate the biological characteristics and prognostic impact of 11q23 deletion and MYCN amplification in patients with bone marrow metastatic neuroblastoma. METHODS: We analyzed the MYCN and 11q23 statuses of 101 patients with bone marrow metastatic neuroblastoma using interphase FISH of bone marrow cells. We specifically compared the biological characteristics and prognostic impact of both aberrations. RESULTS: MYCN amplification and 11q23 deletion were seen in 12 (11.9%) and 40 (39.6%) patients. The two markers were mutually exclusive. MYCN amplification occurred mainly in patients with high lactate dehydrogenase (LDH) and high neuron-specific enolase (NSE) levels (both P < 0.001), and MYCN-amplified patients had more events (tumor relapse, progression, or death) than MYCN-normal patients (P = 0.004). 11q23 deletion was associated only with age (P = 0.001). Patients with MYCN amplification had poorer outcomes than those with normal MYCN (3-year event-free survival [EFS] rate: 8.3 ± 8.0% vs. 43.8 ± 8.5%, P < 0.001; 3-year overall survival [OS] rate: 10.4 ± 9.7% vs. 63.5% ± 5.7%, P < 0.001). 11q23 deletion reflected a poor prognosis only for patients with normal MYCN (3-year EFS rate: 34.3 ± 9.5% vs. 53.4 ± 10.3%, P = 0.037; 3-year OS rate: 42.9 ± 10.4% vs. 75.9 ± 6.1%, P = 0.048). Those with both MYCN amplification and 11q23 deletion had the worst outcome (P < 0.001). CONCLUSIONS: Chromosome band 11q23 deletion predicts poor prognosis only in bone marrow metastatic neuroblastoma patients without MYCN amplification. Combined assessment of the two markers was much superior to single-marker assessment in recognizing the patients at a high risk of disease progression.

Serum Interleukin-6 Level and the rs1800795 Polymorphism in its Gene Associated with Neuroblastoma Risk in Chinese Children.

BACKGROUND: The pro-inflammatory cytokine, interleukin-6 (IL-6), stimulates the metastasis of several neoplasms. An association of its serum level and the single nucleotide polymorphism (SNP) rs1800795 with neuroblastoma (NB) has been reported in American and Italian cohorts. This study was to clarify whether the same association exists in Chinese children. METHODS: A total of 130 NB patients, with 77 boys (59%), 53 girls (41%), mean age 41 ± 5 months, were assigned to two groups: high risk (HR) versus intermediate-low risk (non-HR), and 50 healthy children were randomly selected as the age- and gender-matched controls. Peripheral blood samples were analyzed to determine serum IL-6 level using enzyme linked immunosorbent assay and rs1800795 SNPs phenotype using polymerase chain reaction and gene sequencing. RESULTS: There were 87 NB patients in the HR group and 43 NB patients in the non-HR group. A comparison of allele and genotype frequencies of the rs1800795 polymorphism between patients and controls found no association with NB risk (P > 0.05). The frequency of GG+GC genotype was higher in HR-NB patients than in non-HR-NB patients (64.4% vs. 48.8%, P = 0.02), and serum IL-6 level was much higher in HR-NB patients with GG+GC genotype than in HR-NB patients with CC genotype (4.36 ± 1.1 pg/ml vs. 1.83 ± 0.5 pg/ml; P = 0.02), but not in Non-HR-NB patients. CONCLUSIONS: The polymorphism rs1800795 is associated with serum IL-6 level and level of NB risk. GG genotype might indicate that the tumor is highly malignant (prone to metastasis) and associated with poor prognosis.

Polymorphisms in methotrexate transporters and their relationship to plasma methotrexate levels, toxicity of high-dose methotrexate, and outcome of pediatric acute lymphoblastic leukemia.

High-dose methotrexate (HDMTX) plays an important role in the treatment of acute lymphoblastic leukemia (ALL) although there is great inter-patient variability in the efficacy and toxicity of MTX. The relationship between polymorphisms in genes encoding MTX transporters and MTX response is controversial. In the present study, 322 Chinese children with standard- and intermediate-risk ALL were genotyped for 12 polymorphisms. SLCO1B1 rs10841753 showed a significant association with plasma MTX levels at 48 h (P = 0.017). Patients who had the ABCB1 rs1128503 C allele had longer duration of hospitalization than did those with the TT genotype (P = 0.006). No association was found between oral mucositis and any polymorphism. Long-term outcome was worse in patients with the SLCO1B1 rs4149056 CC genotype than in patients with TT or TC (5-year event-free survival [EFS] 33.3 ± 19.2% vs. 90.5 ± 1.7%, P < 0.001), and was worse in patients with the SCL19A1 rs2838958 AA genotype than in patients with AG or GG (5-year EFS 78.5 ± 4.6% vs. 92.2 ± 1.8%, P = 0.008). Multiple Cox regression analyses revealed associations of minimal residual disease (MRD) at day 33 (hazard ratio 3.458; P = 0.002), MRD at day 78 (hazard ratio 6.330; P = 0.001), SLCO1B1 rs4149056 (hazard ratio 12.242; P < 0.001), and SCL19A1 rs2838958 (hazard ratio 2.324; P = 0.019) with EFS. Our findings show that polymorphisms in genes encoding MTX transporters substantially influence the kinetics and response to HDMTX therapy in childhood ALL.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol.

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

[Progress of studies on genetics of childhood acute leukemia].

This study on determination of leukemia-specific chromosomal abnormalities and their relationship with prognosis of childhood acute leukemia (AL) had an important significance for childhood acute leukemia. In recent years, the efficacy of treatment of childhood AL has been greatly improved, but relapse is still a main factor affecting prognosis. Treatment based on the risk stratification by cytogenetic abnormalities can improve the prognosis and survival rate. In the past 3 decades, the genetic techniques have developed rapidly and many new genetic abnormalities have been found. This review highlights the main chromosomal and genomic abnormalities of 3 common childhood AL, including B-cell precursor acute lymphoblastic leukemia (BCP-ALL), T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML).

[Suppression of NAMPT expression enhances the sensitivity of K562 cells to imatinib and its relative mechanism].

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.