NET1 Enhances Proliferation and Chemoresistance in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most prevalent of pediatric cancers. Neuroepithelial cell-transforming 1 (NET1) has been associated with malignancy in a number of cancers, but the role of NET1 in ALL development is unclear. In the present study, we investigated the effect of NET1 gene in ALL cell proliferation and chemoresistance. We analyzed GEO microarray data comparing bone marrow expression profiles of pediatric B-cell ALL samples and those of age-matched controls. MTT and colony formation assays were performed to analyze cell proliferation. ELISA assays, Western blot analyses, and TUNEL staining were used to detect chemoresistance. We confirmed that NET1 was targeted by miR-206 using Western blot and luciferase reporter assays. We identified NET1 gene as one of the most significantly elevated genes in pediatric B-ALL. MTT and colony formation assays demonstrated that NET1 overexpression increases B-ALL cell proliferation in Nalm-6 cells. ELISA assays, Western blot analyses, and TUNEL staining showed that NET1 contributes to ALL cell doxorubicin resistance, whereas NET1 inhibition reduces resistance. Using the TargetScan database, we found that several microRNAs (miRNAs) were predicted to target NET1, including microRNA-206 (miR- 206), which has been shown to regulate cancer development. To determine whether miR-206 targets NET1 in vitro, we transfected Nalm-6 cells with miR-206 or its inhibitor miR-206-in. Western blot assays showed that miR-206 inhibits NET1 expression and miR-206-in increases NET1 expression. Luciferase assays using wild-type or mutant 3 -untranslated region (3 -UTR) of NET1 confirmed these findings. We ultimately found that miR-206 inhibits B-ALL cell proliferation and chemoresistance induced by NET1. Taken together, our results provide the first evidence that NET1 enhances proliferation and chemoresistance in B-ALL cells and that miR-206 regulates these effects by targeting NET1. This study therefore not only contributes to a greater understanding of the molecular mechanisms underlying B-ALL progression but also opens the possibility for developing curative interventions.     

Lack of Association between Polymorphisms in Genes MTHFR and MDR1 with Risk of Childhood Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is a complex disease caused by interactions betweenhazardous exogenous or/and endogenous agents and many mild effect inherited susceptibility mutations. Someof them are known, but their functional roles still requireinvestigation. Age is a recognized risk factor; childrenwith disease onset after the age of ten have worse prognosis, presumably also triggered by inherited factors.Materials and Methods: The MDR1 gene polymorphisms rs1045642, rs2032582 and MTHFR gene polymorphismsrs1801131 and rs1801133 were genotyped in 68 ALL patients in remission and 102 age and gender matchedcontrols; parental DNA samples were also available for 42 probands. Results: No case control association wasfound between analyzed polymorphisms and a risk of childhood ALL development. Linkage disequilibrium wasnot observed in a family-based association study either. Only marginal association was observed between geneticmarker rs2032582A and later disease onset (p=0.04). Conclusions: Our data suggest that late age of ALL onsetcould be triggered by mild effect common alleles.     

Multidrug Resistance (MDR) Gene Expression in Acute Non Lymphoblastic Leukemia: Sequential Analysis

Sequential evaluation of P-glycoprotein expression was performed in 29 patients with acute nonlymphoblastic leukemia using immunocytochemistry with the C219 antibody. At diagnosis, 32% of the patients exhibited more than 5% of the P-gp(+) leukemic cells. Under chemotherapy, 62% of the patients eventually expressed a subset of P-gp positive leukemic cells. After conventional doses of cytosine-arabinoside (Ara-C) and daunorubicin or mitoxantrone, positive P-gp cells were noted in 65% of the cases. This percentage was significantly higher (p = 0.002) than the proportion of positive cases (15%) observed after regimens containing either intermediate doses of Ara-C or cyclosporine A, a P-gp modulator.     

NOTCH1对T-ALL抑癌基因ID4的负调控作用

目的 研究DNA结合抑制因子4（inhibitor of DNA binding 4, ID4）在急性T淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）中的表达水平,探讨NOTCH1与ID4的相关性及其调控机制。方法 通过Oncomine和Pieters R数据库分析T-ALL患者与正常捐献者中ID4的表达差异及T-ALL患者中NOTCH1与ID4的相关性,用qRT-PCR和Western blot检测阻断NOTCH1或激活NOTCH1后ID4的表达变化进行验证。用生物信息学的方法特异预测出microRNA-342（miR-342）靶向ID4,通过双荧光素酶报告体系进行验证。过表达或沉默miR-342后,qRT-PCR和Western blot检测ID4的表达变化。阻断NOTCH1或激活NOTCH1,qRT-PCR检测miR-342的表达变化。结果 ID4在T-ALL患者中的表达较正常组显著下调（P＜0.01）;ID4受NOTCH1负调控（P＜0.05）;双荧光素酶报告系统验证预测结果正确;过表达miR-342后,ID4的表达受到显著抑制（P＜0.05）,沉默miR-342后,ID4的表达明显上调（P＜0.05）;NOTCH1信号的阻断能够抑制miR-342的表达,NOTCH1信号的激活能够上调miR-342的表达。结论 NOTCH1通过激活miR-342对ID4的负调控作用从而下调抑癌基因ID4的表达,促进T-ALL的发生。     

Relationship between Bax and Bcl-2 Protein Expression and Outcome of Induction Phase Chemotherapy in Pediatric Acute Lymphoblastic Leukemia

Background: Overexpression of the antiapoptotic protein Bcl-2 causes apoptosis to stop and conversely the increased expression of the proapoptotic protein Bax makes lymphoblasts easy to destroy. The Bax/Bcl-2 ratio plays a role in the balance of apoptosis, immortality, resistance, and outcome of chemotherapy. We analyzed the relationship between the Bax/BCl-2 ratio and the outcome of induction phase chemotherapy in pediatric Acute Lymphoblastic Leukemia (ALL). Methods: This research was conducted with a prospective observational study on pediatric ALL aged 1-18 years who were newly diagnosed based on bone marrow aspiration (morphology and immunophenotyping) at Dr. Soetomo General Hospital, Surabaya on October 2020 to March 2021. Expression of Bcl-2, Bax, and Bax/Bcl-2 protein ratio was measured by the flow cytometry method from lymphoblast on bone marrow aspirate samples before and after induction phase chemotherapy according to the 2018 Childhood ALL Indonesian Protocol. The outcomes evaluated were survival and remission rate (lymphoblasts in the bone marrow less than 5%). We used the Mann-Whitney U test and Wilcoxon Signed Rank test to analyze the differences between protein expression with p<0.05 for a two-tailed test. Results: We included 17/26 pediatric ALL, consisting of 88% male, 94% LLA-L1, 76% B cell ALL and 24% T cell ALL. Mean expression of Bax, Bcl-2, and Bax/Bcl-2 protein ratio before chemotherapy among pediatric ALL who alive (N=11) and dead (N=6) were not significantly different (p>0.05). All children who completed the induction phase of chemotherapy went into remission. Bax and Bcl-2 expression before and after chemotherapy showed no difference (p>0.05). The Bax/Bcl-2 ratio increased from 1.74(SD 1.846) to 6.17(4.139) with p=0.021. Conclusion: Expression of Bax, Bcl-2, and Bax/Bcl-2 protein ratio at the beginning of diagnosis did not affect the survival of pediatric ALL after the induction phase of chemotherapy. The Bax/Bcl-2 protein ratio increased 3.5 times in pediatric ALL with remission outcomes, indicating proapoptotic dominance.     

血浆miRNAs在儿童急性淋巴细胞白血病(ALL)不同疾病状态中的表达特点

目的 分析3种血浆miRNA在儿童急性淋巴细胞白血病(ALL)不同疾病状态中的表达特点.方法 选取急性淋巴细胞白血病患儿40例,其中初诊患儿12例,完全缓解患儿16例,复发患儿12例,同时收集我院经健康体检的儿童25例作为正常对照组.抽取所有儿童的空腹静脉血,离心后取血浆,采取直接扩增目的microRNA,并反转录为cDNA,qRT-PCR方法检测血浆miR150、miR155、miR233的表达.结果 ALL组中血浆miR150、miR155、miR233的表达均与正常组有统计学差异;血浆miR150和miR233在初诊组和复发组的表达显著低于缓解组和正常组(P<0.05),血浆miR155在初诊组和复发组的表达显著高于缓解组和正常组(P<0.05).3种miRNA在初诊组与复发组之间、缓解组与正常组之间比较均无统计学差异(P>0.05);初诊组中3种血浆miRNA在不同年龄和性别之间表达均无统计学差异(P>0.05);3种血浆miRNA的AUC(曲线下面积)和95%CI(95%可信区间)均有较好的满意度,且3种血浆miRNA联合检测的精确度更高.结论 血浆miR150、miR155、miR233的表达在儿童急性淋巴细胞白血病(ALL)的诊断、治疗和复发监测中有较高的应用价值.     

TEL/AML1、BCR/ABL、E2A/PBX1、MLL/AF4阳性儿童急性淋巴细胞白血病的临床特点及预后

目的探讨儿童急性淋巴细胞白血病（ALL）TEL/AML1、BCR/ABL、E2A/PBX1、MLL/AF4四种融合基因的表达及其临床诊治意义。方法用实时荧光定量PCR(RQ-PCR)方法检测TEL/AML1、BCR/ABL（p190和p210两种亚型）、E2A/PBX1、MLL/AF4四种融合基因在312例ALL患儿中的表达情况,并总结融合基因阳性患儿的临床和生物学特点、治疗反应及预后情况。结果（1）融合基因阳性共120例,TEL/AML1阳性组72例(23.1%),BCR/ABL阳性组22例(7.1%),p190 16例（5.1%）、p210 6例（1.9%）,E2A/PBX1阳性组18例(5.8%),MLL/AF4阳性组8例(2.6%)。（2）TEL/AML1阳性组诊断时WBC平均值为(18.02±6.45)×109/L,诱导化疗结束完全缓解率(CR)100%,随访期间无复发;BCR/ABL阳性组发病时年龄(9.40±3.55)岁,诱导化疗结束CR 81.8%,强化治疗末仍有6例融合基因未转阴,随访中有8例（36.3%）复发,8例死亡;E2A/PBX1阳性组全部按高危标准给予化疗,诱导化疗结束CR 88.9%,强化治疗期末仍有3例基因微量表达,随访中2例复发;MLL/AF4阳性组发病时WBC(38.41±9.30)×109/L,强化治疗期末仍有2例基因呈阳性,随访中均复发死亡。结论融合基因可作为ALL危险度分层、监测微小残留病、判断预后的重要指标之一。     

Glutathione S-Transferase P1 Genotypes,Genetic Susceptibility and Outcome of Therapy in Thai Childhood Acute Lymphoblastic Leukemia

Glutathione S-transferases (GSTs) are enzymes that involved in bio- transformation by conjugation ofelectrophillic compounds to glutathione. Polymorphisms within genes that encode GSTs may affect the functionof the enzymes. Polymorphisms of GSTP1 at codon 105 residue forms GSTP1 active site for binding of hydrophobicelectrophiles, and the Ile-Val substitution affect substrate specific catalytic activity of this enzyme andmay associate with susceptibility to malignant human disease, especially acute lymphoblastic leukemia (ALL),which is the most common leukemia in children younger than 15 years old.Genetic polymorphisms within theGSTP1 gene of childhood ALL patients were studied. In addition, the association of genetic polymorphism ofGSTP1 and genetic susceptibility of acute lymphoblastic leukemia (ALL) was also determined using Chi-squareand Odds ratio. PCR-RFLP was used to study genetic polymorphism of GSTP1 in 100 ALL patients and 100healthy individuals.The results show that there is no statistically significant association between each genotypesand genetic susceptibility of acute lymphoblastic leukemia (ALL) (OR=0.92, P –value=0.886). Moreover, thereis no statistically significant association between each genotypes and demographic data of acute lymphoblasticleukemia (ALL). However, there are 2 cases of ALL with BM relapse show the polymorphic genotypes of GSTP1.It may suggest that GSTP1*V105 may be involved in relapse of ALL.     

Expression and Long-Term Prognostic Value of CD34 in Childhood and Adult Acute Lymphoblastic Leukemia

Clinical and laboratory characteristics at diagnosis, and long-term (up to 7 years) evolution, were analysed in 156 patients (66 adults and 90 children) with acute lymphoblastic leukemia (ALL), according to the expression of CD34. Eighty-three patients had CD34+ and 73 patients CD34- blasts at diagnosis. In adults, CD34+ ALL was more often of B-lineage (p = .003). White blood cell counts were higher both in children with CD34- B-lineage ALL (p = .02) and adults with CD34- T-lineage ALL (p = .05). Adult patients with CD34- T-lineage ALL were older (p = .04) and had more frequent splenic or liver enlargement (p = .02). Children with CD34+ ALL had a significantly (p = .04) longer event-free follow-up than CD34- children. Survival curves showed that after induction of remission, relapses were less frequent during the first 2 years for patients with CD34+ B-lineage ALL. After 2 years, however, event-free survival curves of CD34+ and CD34- patients merged. Thus it seems that CD34+ has no significant benefit for ALL patients.     

急性白血病患者MUC1与WT1基因表达的临床意义

目的：探讨MUC1基因及WT1基因在急性白血病的表达及临床意义。方法：采用逆转录-聚合酶链反应（RT-PCR）方法，检测54例初发或复发急性白血病（AL）患者MUC1基因以及WT1基因的表达．观察MUC1基因及WT1基因的表达与急性白血病患者疗效关系。结果：54例初治或复发AL中MUC1基因表达阳性率50.0％．WT1基因表达阳性率74．1％。MUC1基因阴性表达的初治非M3型AL 19例治疗完全缓解率达94．7％，MUC1阳性表达的初治非M3型AL 18例治疗完全缓解率55.6％；两组比较有显著性差异（P〈0.05）。WT1基因表达阳性与否与化疗疗效无关。结论：MUC1和WT1基因在急性白血病中有一定的表达，MUC1基因阳性表达可作为急性白血病患者预后不良的一项分子生物学指标。     

MDR1 C3435T and C1236T Polymorphisms: Association with High-risk Childhood Acute Lymphoblastic Leukemia

Background: MDR1, one of the most important drug-transporter genes, encodes P- glycoprotein (P-gp)-atransporter involved in protecting against xenobiotics and multi-drug resistance. The significance of the geneticbackground in childhood acute lymphoblastic leukemia (ALL) is not well understood. Materials and Methods:To evaluate whether C3435T and C1236T MDR1 polymorphisms are associated with the occurrence and outcomeof ALL, 208 children with ALL (median age 5.0 yr) and 101 healthy Thai children were studied by polymerasechain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay. Results: C3435T and C1236TMDR1 polymorphism are significantly associated with the high-risk group (OR= 2.6, 95%CI =1.164-5.808; P=0.028 and OR= 2.231, 95%CI =1.068-4.659; p=0.047, respectively), indicating that both may be candidates formolecular markers in the high-risk group of ALL.     

Expression of MDR-1/P-Glycoprotein in Childhood Acute Megakaryoblastic Leukemia Cells

Multidrug resistance is a major clinical problem in chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often more resistant to many anticancer chemotherapeutic drugs compared to other types of childhood leukemia. There have been reports of the increased expression in hematologic malignancy of multidrug resistant (mdr-I) gene, which encodes for a transmembrane glycoprotein P-glycoprotein that acts as an efflux pump for structurally unrelated chemotherapeutic drugs. We investigated the malignant cells of 15 newly diagnosed childhood AMKL patients by immunocytochemical analysis and found P-glycoprotein expression in all samples from these patients. RNA prepared from five patients at the time of presentation confirmed the expression of mdr-I specific message in all cases by Northern blot analysis. These results imply that malignant cells from all childhood AMKL might express the mdr-I/P-glycoprotein.     

Prognostic factors of childhood acute lymphoblastic leukemia with TCF3::PBX1 in CCCG-ALL-2015: A multicenter study

Background

Contemporary risk-directed treatment has improved the outcome of patients with acute lymphoblastic leukemia (ALL) and TCF3::PBX1 fusion. In this study, the authors seek to identify prognostic factors that can be used to further improve outcome.

Methods

The authors studied 384 patients with this genotype treated on Chinese Children's Cancer Group ALL-2015 protocol between January 1, 2015 and December 31, 2019. All patients provisionally received intensified chemotherapy in the intermediate-risk arm without prophylactic cranial irradiation; those with high minimal residual disease (MRD) ≥1% at day 46 (end) of remission induction were candidates for hematopoietic cell transplantation.

Results

The overall 5-year event-free survival was 84.4% (95% confidence interval [CI], 80.6–88.3) and 5-year overall survival 88.9% (95% CI, 85.5–92.4). Independent factors associated with lower 5-year event-free survival were male sex (80.4%, [95% CI, 74.8–86.4] vs. 88.9%, [95% CI, 84.1–93.9] in female, p = .03) and positive day 46 MRD (≥0.01%) (62.1%, [95% CI, 44.2–87.4] vs. 87.1%, [95% CI, 83.4–90.9] in patients with negative MRD, p < .001). The presence of testicular leukemia at diagnosis (n = 10) was associated with particularly dismal 5-year event-free survival (33.3% [95% CI, 11.6–96.1] vs. 83.0% [95% CI, 77.5–88.9] in the other 192 male patients, p < .001) and was an independent risk factor (hazard ratio [HR], 5.7; [95% CI, 2.2–14.5], p < .001).

Conclusions

These data suggest that the presence of positive MRD after intensive remission induction and testicular leukemia at diagnosis are indicators for new molecular therapeutics or immunotherapy in patients with TCF3::PBX1 ALL.     

Association of Multidrug Resistance Gene-1 (MDR1 C1236T) Polymorphism with the Risk of Acute Myeloid Leukemia in a Moroccan Population

The human multidrug resistance MDR1 gene plays a crucial role in the absorption, transport, metabolism and elimination of harmful compounds. An impaired metabolism of these compounds related to genetic polymorphism may cause cancer such as acute myeloid leukemia AML. Objective: The present study investigated the relationship between C1236T polymorphism and the risk of AML development in a sample of Moroccan population. Methods: The present case-control study included 131 AML patients and 136 healthy controls. The MDR1 C1236T polymorphism was identified by PCR-RFLP method. Meta-analysis was performed to discuss our results. Statistical analyses were performed using SPSS, MetaGenyo and MedCalc. Results: A positive association was found between the 1236TT mutant genotype and the risk of AML (OR 2.39; 95% CI 1.02-5.57, p= 0.04) compared to the wild type 1236CC. In addition, the recessive model revealed that carriers of 1236TT mutant genotype were more exposed to develop AML when compared to the combined 1236CC/CT genotype (OR: 2.27, CI: 1.01–5.05, p=0.04). The clinical parameters of AML showed no significant association. Meta-analysis demonstrated no statistically significant association between this polymorphism and AML susceptibility. Conclusion: Our study suggests that the MDR1C1236T polymorphism appears to be associated with the risk of AML. Further studies, including a large sample size, are needed to confirm these findings.     

Association of lnc-LAMC2-1:1 rs2147578 and CASC8 rs10505477 Polymorphisms with Risk of Childhood Acute Lymphoblastic Leukemia

Long non-coding RNAs (lncRNAs) are a novel class of non-protein coding RNAs that are involved in a wide variety of biological processes. There are limited data regarding the impact of lnc-LAMC2-1:1 rs2147578 as well as CASC8 rs10505477 T>C polymorphisms on cancer development. Here we examined for the first time whether rs2147578 and rs10505477 polymorphisms are associated with childhood acute lymphoblastic leukemia (ALL) in a total of 110 cases and 120 healthy controls. Genotyping was achieved by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The rs2147578 variant increased the risk of ALL in codominant (OR=4.33, 95%CI=2.00-9.37, p<0.0001, CG vs CC, and OR=5.81, 95%CI=2.30-14.69, p=0.0002, GG vs CC), dominant (OR=4.63, 95%CI=2.18-9.86, p<0.0001, CG+GG vs CC), overdominant (OR=1.74, 95%CI=1.02-2.97, p=0.0444, CG vs CC+GG) and allele (OR=1.91, 95%CI=1.32-2.77, p=0.0008, G vs C) inheritance models tested. No significant association was found between the CASC8 rs10505477 T>C variant and risk of childhood ALL. In conclusion, the present study revealed that the lnc-LAMC2-1:1 rs2147578 polymorphism may be a risk factor for developing childhood ALL. Further studies with larger sample sizes with different ethnicities are now required to confirm our findings.     

MDR1 RNA Expression as a Prognostic Factor in Acute Myeloid Leukemia: An Update

In order to confirm our initial report on the negative impact of MDR1 gene expression on the outcome of de novo acute myeloid leukemia (AML), we present an update of our prospective study with a larger number of patients and a longer duration of follow-up. At diagnosis, MDR1 RNA expression of the leukemic cells was negative in 37% and positive in 63% of the patients (N = 79). The complete remission rate of induction chemotherapy was 76% for MDR1 RNA negative and 54% for MDR1 RNA positive patients (p = 0.05). At a median observation duration of 33 months, the duration of overall survival was 19 months for the MDR1 RNA negative patients but only 8 months for the patients with MDR1 gene expression (p = 0.02). Thus the long-term data also indicate that MDR1 gene expression is an unfavourable prognostic factor in AML.