Improving the Identification of High Risk Precursor B Acute Lymphoblastic Leukemia Patients with Earlier Quantification of Minimal Residual Disease

The stratification of patients with acute lymphoblastic leukemia (ALL) into treatment risk groups based on quantification of minimal residual disease (MRD) after induction therapy is now well accepted but the relapse rate of about 20% in intermediate risk patients remains a challenge. The purpose of this study was to further improve stratification by MRD measurement at an earlier stage. MRD was measured in stored day 15 bone marrow samples for pediatric patients enrolled on ANZCHOG ALL8 using Real-time Quantitative PCR to detect immunoglobulin and T-cell receptor gene rearrangements with the same assays used at day 33 and day 79 in the original MRD stratification. MRD levels in bone marrow at day 15 and 33 were highly predictive of outcome in 223 precursor B-ALL patients (log rank Mantel-Cox tests both P<0.001) and identified patients with poor, intermediate and very good outcomes. The combined use of MRD at day 15 (≥1×10⁻²) and day 33 (≥5×1⁻⁵) identified a subgroup of medium risk precursor B-ALL patients as poor MRD responders with 5 year relapse-free survival of 55% compared to 84% for other medium risk patients (log rank Mantel-Cox test, P = 0.0005). Risk stratification of precursor B-ALL but not T-ALL could be improved by using MRD measurement at day 15 and day 33 instead of day 33 and day 79 in similar BFM-based protocols for children with this disease.     

mTOR Inhibition by Everolimus in Childhood Acute Lymphoblastic Leukemia Induces Caspase-Independent Cell Death

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.     

Association between the Polymorphism rs3217927 of CCND2 and the Risk of Childhood Acute Lymphoblastic Leukemia in a Chinese Population

CyclinD proteins, the ultimate recipients of mitogenic and oncogenic signals, play a crucial role in cell-cycle regulation. CyclinD2, one of the cyclinD family, is overexpressed in T-acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia and involved in the pathogenesis of leukemias. Recent reports indicated that CCND2 polymorphisms are associated with human cancer risk, thusly we hypothesized that CCND2 gene polymorphisms may contribute to childhood ALL susceptibility. We selected the polymorphism rs3217927 located in the 3′UTR region of CCND2 to assess its associations with childhood ALL risk in a case-control study. A significant difference was found in the genotype distributions of rs3217927 polymorphism between cases and controls (P = 0.019) and homozygous GG genotype may be an increased risk factor for childhood ALL (adjusted OR  =  1.84, 95% CI  =  1.14 —2.99). Furthermore, this increased risk was more pronounced with GG genotype among high-risk ALL (adjusted OR  = 1.95, 95% CI  =  1.04–3.67), low-risk ALL (adjusted OR  =  2.09, 95% CI  =  1.13–3.87), B-phenotype ALL patients (adjusted OR  =  1.78, 95% CI  =  1.08–2.95) and T-phenotype ALL patients (adjusted OR  =  2.87, 95% CI  =  1.16–7.13). Our results provide evidence that CCND2 polymorphism rs3217927 may be involved in the etiology of childhood ALL, and the GG genotype of rs3217927 may modulate the genetic susceptibility to childhood ALL in the Chinese population. Further functional studies and investigations in larger populations should be conducted to validate our findings.     

Characterization of Fecal Microbiota across Seven Chinese Ethnic Groups by Quantitative Polymerase Chain Reaction

The human gut microbiota consists of complex microbial communities, which possibly play crucial roles in physiological functioning and health maintenance. China has evolved into a multicultural society consisting of the major ethnic group, Han, and 55 official ethnic minority groups. Nowadays, these minority groups inhabit in different Chinese provinces and some of them still keep their unique culture and lifestyle. Currently, only limited data are available on the gut microbiota of these Chinese ethnic groups. In this study, 10 major fecal bacterial groups of 314 healthy individuals from 7 Chinese ethnic origins were enumerated by quantitative polymerase chain reaction. Our data confirmed that the selected bacterial groups were common to all 7 surveyed ethnicities, but the amount of the individual bacterial groups varied to different degree. By principal component and canonical variate analyses of the 314 individuals or the 91 Han subjects, no distinct group clustering pattern was observed. Nevertheless, weak differences were noted between the Han and Zhuang from other ethnic minority groups, and between the Heilongjiang Hans from those of the other provinces. Thus, our results suggest that the ethnic origin may contribute to shaping the human gut microbiota.     

急性B淋巴细胞白血病儿童化疗后微小残留病灶免疫表型改变的分析

目的:分析儿童急性B淋巴细胞白血病(B-cell acute lymphoblastic leukemia,B-ALL)化疗过程中首次微小残留病灶(minimal residual disease,MRD)免疫表型的变化规律及特点,为临床诊断及后续微小残留病的监测提供依据。方法:回顾性分析我院2013年1月至2018年4月收治的393例B-ALL患儿的免疫分型结果及诱导化疗第15天首次MRD数据。结果:(1)在393例白血病中,B-ALL相关特征性免疫表型的出现频率为:CD19+/CD10+/34+64.4%;CD19+/CD10+/34部分表达,15.5%;CD19+/CD34+/CD20+,58.5%;CD19+/CD10+/CD13+,13.2%;CD19+/CD10+/CD33+,5.9%;CD19+/CD10+/CD117+,0.7%;CD19+/CD10+/CD123+,50.1%;CD19+/CD10-/34±,5.9%;CD19+/CD10-/CD20-,2.5%;CD34bright,12.2%;(2)共有285例首次MRD检测结果呈阳性,有181例(63.5%)MRD检测结果至少有1个抗原荧光强度发生改变,其中出现一个抗原强度变化的为83例(29.1%),2个抗原荧光强度变化的为57例(20.0%),3个抗原荧光强度变化为31例(10.9%),4个及4个以上抗原荧光强度变化为10例(3.5%)。抗原荧光强度变化频率最高的依次为CD45、CD34、CD20;(3)共有7例患儿复发,复发时行免疫分型检测,其中4例与初发时有抗原荧光强度变化。结论:(1)儿童B-ALL远高于其他白血病类型,且具有独特的相关特征性免疫表型。初发B-ALL免疫分型结果不仅可完善白血病MICM分型,更是化疗后MRD监测的线索及客观依据;(2)在儿童B-ALL化疗过程中,免疫表型极有可能会发生变化,在后续的微小残留病灶检测过程中应注意对变化抗原的判断和追踪。     

Maternal Benzene Exposure during Pregnancy and Risk of Childhood Acute Lymphoblastic Leukemia: A Meta-Analysis of Epidemiologic Studies

Background

The prevalence of childhood leukemia is increasing rapidly all over the world. However, studies on maternal benzene exposure during pregnancy and childhood acute lymphoblastic leukemia (ALL) have not been systematically assessed. Therefore, we performed a meta-analysis to investigate the association between maternal solvent, paint, petroleum exposure, and smoking during pregnancy and risk of childhood ALL.

Methods

Relevant studies up to September 1^st, 2013 were identified by searching the PubMed, EMBASE, Cochrane library and the Web of Science databases. The effects were pooled using either fixed or random effect models based on the heterogeneity of the studies.

Results

Twenty-eight case-control studies and one cohort study were included for analysis, with a total of 16,695 cases and 1,472,786 controls involved. Pooled odds ratio (OR) with 95% confidence interval (CI) for ALL was 1.25 (1.09, 1.45) for solvent, 1.23 (1.02, 1.47) for paint, 1.42 (1.10, 1.84) for petroleum exposure, and 0.99 (0.93, 1.06) for maternal smoking during pregnancy. No publication bias was found in this meta-analysis and consistent results were observed for subgroup and sensitivity analyses.

Conclusions

Childhood ALL was associated with maternal solvent, paint, and petroleum exposure during pregnancy. No association was found between ALL and maternal smoking during pregnancy. Avoidance of maternal occupational and environmental benzene exposure during pregnancy could contribute to a decrease in the risk of childhood ALL.     

PCR法快速检测临床标本中结核杆菌DNA

应用聚合酶链反应（ＰＣＲ）快速检测临床标本（脑脊液、胸水、腹水、血、痰液）中的结核杆菌ＤＮＡ,特异性扩增片段１２３ｂｐ,为结核杆菌的特异性重复序列ＩＳ６１１０部分基因。ＰＣＲ检测人型结核杆菌的敏感性达１０ｆｇＤＮＡ。临床标本的ＰＣＲ检测阳性率（２３．３％）明显高于抗酸染色涂片（２．９％）和细菌培养（５．７％）的阳性率（Ｐ〈０．０５）。通过设立对照系统及对扩增产物酶切分析,表明该法无假阴性结果（特异     

Using a Bayesian Hierarchical Model for Identifying Single Nucleotide Polymorphisms Associated with Childhood Acute Lymphoblastic Leukemia Risk in Case-Parent Triads

Childhood acute lymphoblastic leukemia (ALL) is a condition that arises from complex etiologies. The absence of consistent environmental risk factors and the presence of modest familial associations suggest ALL is a complex trait with an underlying genetic component. The identification of genetic factors associated with disease is complicated by complex genetic covariance structures and multiple testing issues. Both issues can be resolved with appropriate Bayesian variable selection methods. The present study was undertaken to extend our hierarchical Bayesian model for case-parent triads to incorporate single nucleotide polymorphisms (SNPs) and incorporate the biological grouping of SNPs within genes. Based on previous evidence that genetic variation in the folate metabolic pathway influences ALL risk, we evaluated 128 tagging SNPs in 16 folate metabolic genes among 118 ALL case-parent triads recruited from the Texas Children’s Cancer Center (Houston, TX) between 2003 and 2010. We used stochastic search gene suggestion (SSGS) in hierarchical Bayesian models to evaluate the association between folate metabolic SNPs and ALL. Using Bayes factors among these variants in childhood ALL case-parent triads, two SNPs were identified with a Bayes factor greater than 1. There was evidence that the minor alleles of NOS3 rs3918186 (OR = 2.16; 95% CI: 1.51-3.15) and SLC19A1 rs1051266 (OR = 2.07; 95% CI: 1.25-3.46) were positively associated with childhood ALL. Our findings are suggestive of the role of inherited genetic variation in the folate metabolic pathway on childhood ALL risk, and they also suggest the utility of Bayesian variable selection methods in the context of case-parent triads for evaluating the role of SNPs on disease risk.     

人单拷贝及重复DNA序列的原位PCR

在培养的人小肠癌转移腹水细胞系细胞中进行了Y染色体特异的重复序列及单拷贝序列的原位扩增与检测.结果显示原位PCR法的灵敏度比直接的原位杂交法明显提高.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒(CAV)环形基因组DNA(全长2.3kb)的EcoRI位点和BamHI位点的两侧选择适当序列合成两对引物,用PCR技术,从斑点杂交检测到病毒核酸的CAV感染的MDCC-RP1细胞基因组DNA中,分别扩增出包含EcoRI和BamHI分割开的病毒基因组两部分(1.5kb和0.8kb)约1.5kb和约1.25kb的两个片段.再将其中相应序列拼接克隆进pUC18载体,获得包含CAV全基因组序列DNA片段的克隆质粒pCAV2.4.酶切分析表明,该质粒具有预期的BamHI位点、PstI位点、HindⅢ位点,而预期的EcoRI位点消失.重组质粒插入DNA片段的两端序列分析表明,质粒pCAV2.4是包含CAV全基因组序列的重组质粒,插入DNA片段序列中的EcoRI位点序列发生了一个碱基突变.     

Detection of Human Cytomegalovirus DNA in Breast Milk by Means of Polymerase Chain Reaction

Three hundred and twenty-five breast milk samples were examined for the occurrence of human cytomegalovirus (HCMV) by cell culture method. Virus was isolated from the milk in 1 of 177 samples collected within 6 days after delivery, 2 of 115 samples collected during the period of 7 days to 1 month after delivery, 10 of 33 samples collected over 1 month after delivery. Next, we tried to amplify HCMV DNA from the breast milk samples from HCMV seropositive mothers and seronegative mothers at 1 month after delivery by polymerase chain reaction. HCMV DNA was detected in 12 of 13 samples from seropositive mothers and in none of 7 samples from seronegative mothers. It was thought that all women seropositive for HCMV principally shed the virus into their breast milk at 1 month after delivery.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Summary Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10³ copies ml^-1 in all samples and 1.0 × 10⁴ copies ml^--1 in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10³ copies ml^--1, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.     

Extremely Low-Frequency Magnetic Fields and the Risk of Childhood B-Lineage Acute Lymphoblastic Leukemia in a City With High Incidence of Leukemia and Elevated Exposure to ELF Magnetic Fields

It is important to study the relationship between extremely low-frequency magnetic fields (ELF-MFs) and childhood leukemia, particularly in locations with a high incidence of this neoplasm in children and an elevated exposure to ELF-MF, such as Mexico City. The aim was to investigate the association between ELF-MF exposure and the risk of B-lineage acute lymphoblastic leukemia (B-ALL). A case–control study was conducted in Mexico City during the period from 2010 to 2011. Residential 24-h ELF-MF measurements were obtained for 290 incident B-ALL patients and 407 controls, aged less than 16 years. Controls were frequency-matched by sex, age (±18 months), and health institution. The adjusted odds ratios (aOR) and 95% confidence intervals (CIs) were calculated. ELF-MF exposure at <0.2 μT was used to define the reference group. ELF-MF exposure at ≥0.3 μT was observed in 11.3% of the controls. Different ELF-MF intensity cutoff values were used to define the highest exposure category; the highest exposure category for each cutoff value was associated with an increased risk of B-ALL compared with the corresponding lower exposure categories. The aORs were as follows: ≥0.2 μT = 1.26 (95% CI: 0.84–1.89); ≥0.3 μT = 1.53 (95% CI: 0.95–2.48); ≥0.4 μT = 1.87 (95% CI: 1.04–3.35); ≥0.5 μT = 1.80 (95% CI 0.95–3.44); ≥0.6 μT = 2.32 (95% CI: 1.10–4.93). ELF-MF exposure as a continuous variable (per 0.2 μT intervals) was associated with B-ALL risk (aOR = 1.06; 95% CI: 1.01–1.12). In the present study, the proportion of children exposed to ≥0.3 μT is among the highest reported worldwide. Additionally, an ELF-MF exposure ≥0.4 μT may be associated with the risk of B-ALL. Bioelectromagnetics. © 2020 Bioelectromagnetics Society     

Roles of Genetic Polymorphisms in the Folate Pathway in Childhood Acute Lymphoblastic Leukemia Evaluated by Bayesian Relevance and Effect Size Analysis

In this study we investigated whether polymorphisms in the folate pathway influenced the risk of childhood acute lymphoblastic leukemia (ALL) or the survival rate of the patients. For this we selected and genotyped 67 SNPs in 15 genes in the folate pathway in 543 children with ALL and 529 controls. The results were evaluated by gender adjusted logistic regression and by the Bayesian network based Bayesian multilevel analysis of relevance (BN-BMLA) methods. Bayesian structure based odds ratios for the relevant variables and interactions were also calculated. Altogether 9 SNPs in 8 genes were associated with altered susceptibility to ALL. After correction for multiple testing, two associations remained significant. The genotype distribution of the MTHFD1 rs1076991 differed significantly between the ALL and control population. Analyzing the subtypes of the disease the GG genotype increased only the risk of B-cell ALL (p = 3.52×10⁻⁴; OR = 2.00). The GG genotype of the rs3776455 SNP in the MTRR gene was associated with a significantly reduced risk to ALL (p = 1.21×10⁻³; OR = 0.55), which resulted mainly from the reduced risk to B-cell and hyperdiploid-ALL. The TC genotype of the rs9909104 SNP in the SHMT1 gene was associated with a lower survival rate comparing it to the TT genotype (80.2% vs. 88.8%; p = 0.01). The BN-BMLA confirmed the main findings of the frequentist-based analysis and showed structural interactional maps and the probabilities of the different structural association types of the relevant SNPs especially in the hyperdiploid-ALL, involving additional SNPs in genes like TYMS, DHFR and GGH. We also investigated the statistical interactions and redundancies using structural model properties. These results gave further evidence that polymorphisms in the folate pathway could influence the ALL risk and the effectiveness of the therapy. It was also shown that in gene association studies the BN-BMLA could be a useful supplementary to the traditional frequentist-based statistical method.