FOXO3 gene polymorphisms influence the risk of acute lymphoblastic leukemia in Chinese children

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed cancer in children and single-nucleotide polymorphisms (SNPs) in certain genes influence risk of ALL. Although FOXO3 had been demonstrated to be involved leukemia, the role of FOXO3 polymorphisms was still not clear. In the present study, we explored the association of FOXO3 SNPs with ALL risk in Chinese children. We genotyped four polymorphisms (rs17069665 A>G, rs4945816 T>C, rs4946936 C>T, and rs9400241 A>C) of FOXO3 in 425 ALL cases and 1339 health controls. The associations were estimated by odds ratios (ORs) with their 95% confidence intervals (CIs). Further analyses were performed to explore associations of rs17069665 and rs9400241 with ALL susceptibility in terms of age, gender, immunophenotype, minimal residual disease (MRD), and other clinical characteristics. We found rs17069665 related to the increased ALL risk (OR = 1.76; 95% CI = 1.02-3.04), rs9400241 related to decreased ALL risk (OR = 0.80; 95% CI = 0.64-0.99). The effects of rs17069665 on ALL risk were more predominant in males and children < 10 years, and patients with lower rates of platelet or neutrophil. As for rs9400241, the effects were more predominant in children < 10 years, and in patients with pre B ALL, positive MRD, anemia, or hepatomegaly. In conclusion, FOXO3 gene polymorphisms influence the risk of ALL in children and might be a potential biomarker for ALL susceptibility.     

Microtubules play an essential role in the survival of primary acute lymphoblastic leukemia cells advancing through G1 phase

We recently reported that primary acute lymphoblastic leukemia (ALL) cells are susceptible to the microtubule depolymerizing agent vincristine (VCR) in G1 phase. This finding prompted testing another G1 phase-active compound, palbociclib (PCB), a highly selective inhibitor of cyclin-dependent kinases 4/6 (CDK4/6), alone and in combination with VCR. PCB used alone caused G1 arrest in ALL cells with no effect on cell viability, and similar results were obtained for the retinoblastoma (RB)-proficient T98G glioblastoma cell line. In contrast, HeLa cells failed to arrest in the presence of PCB, consistent with their lack of dependence on the CDK4/6-RB pathway. When ALL cells were pretreated with PCB, they became refractory to death in G1 phase induced by VCR treatment, whereas HeLa cells retained VCR sensitivity after PCB pretreatment. Immunofluorescence microscopy showed that PCB did not disrupt the microtubule network nor prevent VCR from doing so. Furthermore, ALL cells pretreated with PCB retained susceptibility to the Bcl-2/Bcl-xL inhibitor ABT-263, indicating that downstream apoptotic signaling was unaffected. When released from PCB-enforced arrest, ALL cells reinitiated cycling and regained sensitivity to VCR. ALL cells treated with cycloheximide also arrested in G1 phase and became insensitive to VCR, independently reinforcing conclusions derived from PCB-imposed arrest. Thus, primary ALL cells advancing through G1 phase are strictly dependent on functional microtubules for survival whereas microtubules are dispensable for G1-arrested cells. These findings provide novel insight into interphase microtubule function and, from a therapy standpoint, strongly caution against combining microtubule targeting agents and CDK4/6 inhibitors for ALL.     

The complex translocation (9;14;14) involving IGH and CEBPE genes suggests a new subgroup in B-lineage acute lymphoblastic leukemia

Many subtypes of acute lymphoblastic leukemia (ALL) are associated with specific chromosomal rearrangements. The complex translocation t(9;14;14), a variant of the translocation (14;14)(q11;q32), is a rare but recurrent chromosomal abnormality involving the immunoglobulin heavy-chain (IGH) and CCAAT enhancer-binding protein (CEBPE) genes in B-lineage ALL (B-ALL) and may represent a new B-ALL subgroup. We report here the case of a 5-year-old girl with B-ALL, positive for CD19, CD38 and HLA-DR. A direct technique and G-banding were used for chromosomal analysis and fluorescentin situ hybridization (FISH) with BAC probes was used to investigate a possible rearrangement of the IGH andCEBPE genes. The karyotype exhibit the chromosomal aberration 46,XX,del(9)(p21),t(14;14)(q11;q32). FISH with dual-color break-apartIGH-specific and CEPBE-specific bacterial artificial chromosome (BAC) probes showed a complex t(9;14;14) associated with a deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) and paired box gene 5 (PAX5) at 9p21-13 and duplication of the fusion gene IGH-CEBPE.     

miRNA-155 and miRNA-181a as prognostic biomarkers for pediatric acute lymphoblastic leukemia

Mortality rates of acute lymphoblastic leukemia (ALL) have improved over the past 20 years; however, a significant portion of deaths stems from the lack of prognostic biomarkers, which can direct therapy and overcome drug resistance. microRNA-155a (miRNA-155a) and miRNA-181a are two single-stranded miRNAs involved in the pathogenesis of many types of leukemia and lymphoma and is linked to drug resistance. We investigated their expression levels in 55 patients, 45 diagnosed with ALL and 10 as a control group. We found that miRNA-155a and miRNA-181a were significantly upregulated in the ALL group with both being linked to high levels of minimal residual disease and poor prognosis. miRNA-155a cutoff value was significant in discriminating between high- and low-risk ALL patients as well as between ALL patients and healthy controls, miRNA-181a cutoff value, however, was not significant. Both markers levels were significantly downregulated after therapy. We conclude that miR-155 is correlated with poor prognosis in ALL, whereas we couldn’t link miRNA-181a to the prognosis in ALL. Moreover, the marked decrease in their expression after therapy could reflect their impact on disease outcome.     

Fabrication of Au/Fe3O4/RGO based aptasensor for measurement of miRNA‐128, a biomarker for acute lymphoblastic leukemia (ALL)

Due to their high sensitivity, simplicity, portability, self‐contained, and low cost, the development of electrochemical biosensors is a beneficial way to diagnose and anticipate many types of cancers. An electrochemical nanocomposite‐based aptasensor is fabricated for the determination of miRNA‐128 concentration as the acute lymphoblastic leukemia (ALL) biomarker for the first time. The aptamer chains were immobilized on the surface of the glassy carbon electrode (GCE) through gold nanoparticles/magnetite/reduced graphene oxide (AuNPs/Fe₃O₄/RGO). Fast Fourier transform infrared (FTIR), X‐ray diffraction (XRD), vibrating sample magnetometer (VSM), and transmission electron microscopy (TEM) were used to characterize synthesized nanomaterials. Cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) were used to characterize the modified GCE in both label‐free and labeled methods. The results indicate that the modified working electrode has high selectivity and for miRNA‐128 over other biomolecules. The hexacyanoferrate redox system typically operated at around 0.3 V (vs. Ag/AgCl), and the methylene blue redox system ran at about 0 V, were used as an electrochemical probe. The detection limit and linear detection range for hexacyanoferrate and methylene blue are 0.05346 fM, 0.1–0.9 fM, and 0.005483 fM, 0.01–0.09 fM, respectively. The stability and diffusion control analyses were performed as well. In both label‐free and labeled methods, the modified electron showed high selectivity for miRNA‐128. The use of methylene blue as a safer redox mediator caused miRNA‐128 to be detected with greater accuracy at low potentials in PBS media. The findings also show the substantial improvement in detection limit and linearity by using reduced graphene oxide‐magnetite‐gold nanoparticles that can be verified by comparing with previous studies on the detection of other miRNAs.     

Cytokine and cellular responses to human herpesvirus‐6B in patients with B‐acute lymphoblastic leukemia

Primary infection with human herpesvirus‐6 (HHV‐6), is followed by its lifelong persistence in the host. Most T‐cell responses to HHV‐6 have been characterized using peripheral blood from healthy adults; however, the role of HHV‐6 infection in immune modulation has not been elucidated for some diseases. Therefore, in this study the immune response to HHV‐6 infection in patients with B‐acute lymphoblastic leukemia (B‐ALL) was analyzed. HHV‐6 load was quantified in blood samples taken at the time of diagnosis of leukemia and on remission. The same concentrations of anti‐ and pro‐inflammatory cytokines (IL‐4, IL‐1, IL‐6, IL‐8, IL‐12p70, IL‐17a, TNF‐α and IFN‐γ) were detected in plasma samples from 20 patients with and 20 without detectable HHV‐6 virus loads in blood. Characterization of T‐cell responses to HHV‐6 showed low specific T‐cells frequencies of 2.08% and 1.46% in patients with and without detectable viral loads, respectively. IFN‐γ‐producing T cells were detected in 0.03%–0.23% and in 0%–0.2% of CD4+T cells, respectively. Strong production of IL‐6 was detected in medium supernatants of challenged T‐cells whatever the HHV‐6 status of the patients (973.51 ± 210.06 versus 825.70 ± 210.81 pg/mL). However, concentrations of TNF‐α and IFN‐γ were low. Thus, no association between plasma concentrations of cytokines and detection of HHV‐6 in blood was identified, suggesting that HHV‐6 is not strongly associated with development of B‐ALL. The low viral loads detected may correspond with latently infected cells. Alternatively, HHV‐6B specific immune responses may be below the detection threshold of the assays used.     

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.     

Asparaginase (native ASNase or pegylated ASNase) in the treatment of acute lymphoblastic leukemia

The discovery of the tumor-inhibitory properties of asparaginase (ASNase) began in the early 1950s with the observation that guinea pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. About 4000 cases of acute lymphoblastic leukemia (ALL) are diagnosed very year in the US and many more through out the world. The majority of these cases are in children and young adults, making ALL the most common form of malignancy in these age groups. The treatment protocols of ALL are complex and use 6-12 drugs. Consequently, the improvement in the protocol design has improved significantly the success rate for long-term event-free survival in the past 20-30 years, which is now approximately 75% for patients afflicted with the higher risk ALL features and just above this percentage for patients with standard or good features. Despite this success, approximately 15% of patients die from ALL, making leukemic relapse the most common cause of treatment failure in pediatric oncology. ASNases have been the cornerstone of ALL therapies since the late 1970s. Native or pegylated L-asparaginase (ASNase or PEG-ASNase) are highly specific for the deamination of L-asparagine (Asn) to aspartic acid and ammonia. Depletion of Asn leads to a nutritional deprivation and inhibition of protein biosynthesis, resulting in apoptosis in T-lymphoblastic leukemias, which require Asn from external sources. The reactions of the host exposed to repeated ASNase treatments as well as the up-regulation of the mammalian enzymes to overcome the ASN-depletion toxic condition are of significant importance and may make us relearn the lessons on this important antileukemic drug.     

GX15-070 (obatoclax) overcomes glucocorticoid resistance in acute lymphoblastic leukemia through induction of apoptosis and autophagy

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies including acute lymphoblastic leukemia (ALL). The BCL-2 family has an essential role in regulating GC-induced cell death. Here we show that downregulation of antiapoptotic BCL-2 family proteins, especially MCL-1, enhances GC-induced cell death. Thus we target MCL-1 by using GX15-070 (obatoclax) in ALL cells. Treatment with GX15-070 in both dexamethasone (Dex)-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases the Annexin V-positive population, which is indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from the BAK/MCL-1 complex following GX15-070 treatment. Consistently, downregulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, downregulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Downregulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy.     

Changes of CSF-Cu and -Zn in children with acute lymphoblastic leukemia

In 20 Dutch children with acute lymphoblastic leukemia (ALL), Cu and Zn levels in cerebrospinal fluid (CSF) were studied during standard treatment (Protocol ALL-BFM-86/SNWLK-ALL-VII). CSF-Cu in 10 controls was 0.04±0.02 μmol/L, lower compared to values in adults. At the moment of diagnosis, CSF-Cu values were higher, 0.06±0.03 μmol/L, and during maintenance therapy lower, 0.01±0.01 μmol/L. Children with central nervous system (CNS) involvement ALL as judged by CAT Scan and EEG—in addition to cytology—showed lower CSF-Cu values compared to children without. CSF-Zn values were also measured. CSF-Zn was 0.05 μmol/L and did not vary. Cu/Zn molar ratios were increased at the onset of treatment, and decreased during maintenance therapy. The changes in CSF-Cu may follow the natural course of the disease or may relate to the success of treatment, reflecting a decrease of leukemia activity. Another explanation concerns a risk to CNS damage by low CSF-Cu causing neuron dysfunction. Conditions necessary for the interpretation of these results into a clinical strategy for followup study are outlined.     

Overexpression of heme oxygenase-1 in microenvironment mediates vincristine resistance of B-cell acute lymphoblastic leukemia by promoting vascular endothelial growth factor secretion

Chemoresistance often causes treatment failure of B-cell acute lymphoblastic leukemia (B-ALL). However, the mechanism remains unclear at present. Herein, overexpression of heme oxygenase-1 (HO-1) was found in the bone marrow stromal cells (BMSCs) from B-ALL patients developing resistance to vincristine (VCR), a chemotherapeutic agent. Two B-ALL cell lines Super B15 and CCRF-SB were cocultured with BMSCs transfected with lentivirus to regulate the expression of HO-1. Silencing HO-1 expression in BMSCs increased the apoptotic rates of B-ALL cell lines induced by VCR, whereas upregulating HO-1 expression reduced the rate. Cell cycle can be arrested in the G2/M phase by VCR. In contrast, B-ALL cells were arrested in the G0/G1 phase due to HO-1 overexpression in BMSCs, which avoided damage from the G2/M phase. Vascular endothelial growth factor (VEGF) in BMSCs, as a key factor in the microenvironment-associated chemoresistance, was also positively coexpressed with HO-1. VEGF secretion was markedly increased in BMSCs with HO-1 upregulation but decreased in BMSCs with HO-1 silencing. B-ALL cell lines became resistant to VCR when cultured with VEGF recombinant protein, so VEGF secretion induced by HO-1 expression may promote the VCR resistance of B-ALL cells. As to the molecular mechanism, the PI3K/AKT pathway mediated regulation of VEGF by HO-1. In conclusion, this study clarifies a mechanism by which B-ALL is induced to resist VCR through HO-1 overexpression in BMSCs, and provides a novel strategy for overcoming VCR resistance in clinical practice.     

XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5’ cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.     

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia

 Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor α-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone, CD25 expression was significantly suppressed in CD4⁺ T cells from 27 of 48 (56.3%) cases and in CD8⁺ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells, CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal. Received: 27 March 1996 / Accepted: 23 December 1996     

Association of the macrophage migration inhibitory factor gene − 173G/C polymorphism with inflammatory bowel disease: A meta-analysis of 4296 subjects

A variety of epidemiologic studies have focused on the association between macrophage migration inhibitory factor (MIF) gene − 173G/C polymorphism and inflammatory bowel disease (IBD). However, results in different studies have been inconsistent. In order to derive a more precise estimation of the associations, we performed this meta-analysis and systematic searches of electronic databases PubMed and Web of Science (up to April 30, 2013). Based on our search criteria, a total of seven eligible studies concerning the MIF − 173G/C polymorphism and IBD risk were included in the final meta-analysis, comprising 2162 IBD cases and 2134 controls. Significant association was found between MIF − 173G/C polymorphism and the risk of IBD when all studies were pooled into the meta-analysis (for C allele vs. G allele: OR = 1.25, 95% CI = 1.12–1.41, p = 0.000; for C/C vs. G/G: OR = 1.71, 95% CI = 1.23–2.39, p = 0.002; for C/C + G/C vs. G/G: OR = 1.24, 95% CI = 1.09–1.42, p = 0.002; for C/C vs. G/C + G/G: OR = 1.67, 95% CI = 1.20–2.33, p = 0.002). Heterogeneity and publication bias did not exist in the overall comparisons. The present meta-analysis suggests an association between the MIF − 173G/C polymorphism and IBD risk. However, due to few studies and the selection bias existed in some studies, the results should be interpreted with caution.     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.