Clinical Implication of Toll-Like Receptors (TLR2 and TLR4) Polymorphisms in Adult Patients with Acute B-cell Lymphoblastic Leukemia

Backgrounds: Neutropenia after intensive chemotherapy of acute lymphoblastic leukemia (ALL) could lead to infectious complications that affect outcome of acute leukemia patients. Many single-nucleotide polymorphisms (SNPs) of Toll-like receptors (TLRs) can affect the genetic susceptibility to infections. We investigated the impact of different SNPs on the incidence of developing sepsis and pneumonia in patients with newly diagnosed B-ALL following induction chemotherapy. Subjects and methods: We analyzed three SNPs in the TLR2 (Arg753Gln) and TLR4 (Asp299Gly& Thr399Ile) genes using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) in a case control study of 40 precursor B-ALL patients and 50 control subjects. The risk of developing sepsis and pneumonia were assessed by multiple logistic regression analyses. Results: The presence of the TLR-2 AG polymorphism was significantly associated with pneumonia in B-ALL patients. Furthermore, TLR4 Thr399Ile AG was a risk factor for sepsis in B-ALL patients. Moreover; Significant association between TLR-2 AA, TLR-4 CC and TL-4 AA genotypes and longer OS were detected in studied B-ALL patients. Conclusion: We concluded that TLR-4 (AG and CT) genotypes are associated with high susceptibility to sepsis and pneumonia respectively; while, TLR-2, TLR-4 AA and TLR-4 CC genotypes  could predict good B-ALL patients outcome.     

Defining Acute Myeloid Leukemia Ontogeny in Older Patients

BackgroundAcute myeloid leukemia (AML) in elderly patients is associated with poor outcomes and often arises from antecedent hematologic disorders (AHD), classified as secondary AML (sAML).Patients and MethodsTo validate the use of somatic mutations to determine AML ontogeny in the elderly population, we identified 178 elderly (> 70 years) patients with AML with NexGen Sequencing data. Patients were divided clinically into primary AML (pAML) or sAML based on prior history of AHD. Patients were then reclassified into 4 groups based on somatic mutations and cytogenetics as suggested by Lindsley et al: group 1 (pAML) with CBF rearrangements, 11q23/MLL, and NPM1 mutation (MT); group 2 (sAML) with SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 MT; group 3 with TP53 MT; and group 4 as not otherwise specified (NOS).ResultsBased on clinical criteria, 95 patients were classified as pAML and 82 patients as sAML. Based on the AML ontogeny proposed, 8 patients were classified as pAML, 72 patients as sAML, 28 patients had TP53 MT, and 70 patients were classified as NOS. The median overall survival was 22.4,14, 2.8, and 11.2 months, respectively. Clinical versus molecular classification was discordant where 25% (n = 2) of patients classified as pAML by molecular signature had a history of AHD, whereas 44% (n = 32) of patients classified molecularly as sAML had no prior AHD. In the TP53 MT and NOS categories, 37% (n = 28) and 43% (n = 70) of patients had AHD, respectively.ConclusionOur data shows that molecular annotation of elderly patients with AML reclassifies a significant proportion of patients as sAML, which may have therapeutic implications.     

Clinical Implication of DNMT3A and TET2 Genes Mutations in Cytogenetically Normal Acute Myeloid Leukemia

Background: Refining risk stratification of cytogenetically  normal AML (CN-AML) cases is important for decision making and tailoring of therapy. In this context genetic and epigenetic mutations was considered. Among these epigenetic regulators are DNMT3A & TET2 genes. Therefore, the aim of  this study was to determine the prevalence of DNMT3A and TET2 genes mutations and their impact on the outcome of  adult AML patients. Subjects and methods: The present study is cross sectional study which was conducted on 39 adult CN-AML patients at diagnosis. For all included patients sanger sequencing was done for DNMT3A exon 23 and TET2 exon 3 genes. Results: DNMT3A mutations were detected in 8 of 39 patients (20.5%), and in 5 of 39 patients(12.8%) in TET gene. Two CN-AML  patients had combined mutations in both genes. All of the mutations detected were missense and only one was frame shift. Mutated TET2 or DNMT3A genes were significantly associated with failure of complete remission (CR) (p <0.001), higher mortality rate, shorter OS (mean=16 versus 22.7 months) and shorter DFS (mean= 9.5 versus 21.4 months) when compared to non-mutated ones. Conclusion: Mutated TET2 and DNMT3A detection define a subgroup of CN-AML patients with poor outcome.     

Expression of Enhancer of Zeste Homolog 2 (EZH2) Gene in Acute Myeloid Leukemia

Background: Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of the chromatin modifying enzyme polycomb repressive complex 2 (PRC2). As a complex, these proteins selectively silence target genes through trimethylation of histone 3 at lysine 27. EZH2 is strongly oncogenic. It has been observed in various malignancies which makes it an interesting therapeutic target. Whether it functions as a tumor suppressor or oncogene in acute leukemia is not settled. Aim of this work: This study aimed at determining the expression levels of EZH2 gene in a cohort of adult Egyptian patients newly diagnosed with acute myeloid leukemia (AML). Materials and methods: The present study included 45 de novo AML patients and 40 healthy subjects of matched age and sex as a control group. All study participants were subjected to complete blood count (CBC), bone marrow examination, immunophenotyping, conventional cytogenetic studies and Detection of EZH2 gene expression levels by real time quantitative polymerase chain reaction (RQ-PCR). Results: EZH2 was significantly downregulated in AML patients compared to controls (p<0.001). There was no significant difference in EZH2 level when considering age, sex, bone marrow blasts count, cytogenetic studies, type or site of infection. Low EZH2 expression was associated with higher mortality (31 patients, 68.9%). Conclusions: Low EZH2 expression is prevalent in Egyptian AML patients subsequently; it is suggested to function as tumor suppressor gene rather than an oncogene. Moreover, EZH2 downregulation is associated with resistance to chemotherapy and high mortality rate.     

DNA Repair Genes Polymorphisms: Impact on Acute Myeloid Leukemia Patients Outcome

Background: ATM; XRCC6 and LIG4 genes play an important role in repairing the double-strand DNA breaks and maintaining the genome stability. Single nucleotide polymorphisms (SNPs) in these genes could affect these genes expression and function. The aim of this study was to address the effect of SNP of the DNA repairing genes on corresponding  gene expression as well as AML patient’s outcome. Subjects and Methods: This is cross sectional study included 95 newly diagnosed AML patients. For all subjects included in our study SNPs  and expression of ATM (rs189037G>A), XRCC6 (rs2267437C>G) and LIG4 (rs1805388C>T) genes were evaluated by RFLP and real time PCR. Results:The following SNPs in ATM (AA); XRCC6 (GG); and LIG4 (TT) are associated with down regulation of the corresponding genes (P<0.001). The lower expression of ATM and LIG4 genes are associated with shorter OS and DFS. Cox regression multivariate analysis revealed that lower expression of ATM HR : 2.02 (CI: 1.12-3.64; p=0.020. Conclusion: The following SNPs of ATM (AA); XRCC6 (GG); and LIG4 (TT) are associated with down regulation of corresponding genes expression. ATM and XRCC6 lower expression are predictors of OS while ATM is predictor of DFS and could be used for optimizing the AML therapy.     

Association of SDF-1 Gene Polymorphism with Increased Risk of Acute Myeloid Leukemia Patients

Background: Acute myeloid leukemia (AML) is a heterogenous group of disorders that emerge from the malignant transformation of hematopoietic stem cells. Chemokine stromal cell-derived factor 1(SDF-1) and its receptor CXC receptor 4 (CXCR4) has an essential role in dissemination of blast cells. Study aimed to detect CXCR4 expression and the SDF-1 (rs1801157) gene polymorphisms and correlate them with prognosis and outcome in AML patients. Subjects and Methods: The study was conducted on 60 de-novo AML patients, and 60 healthy controls. SDF-1 (rs1801157) gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and CXCR4 expression was done using flow cytometry analysis. Results: SDF-1 dominant model (AG+AA) had higher risk AML (p 0.002). CXCR4positive cases were associated significantly with toxic manifestations (p 0.019), lower CR rates (p 0.004), and unfavorable cytogenetics (p 0.027). Multivariate analysis showed that combined CXCR4positive with dominant SDF-1 considered as independent prognostic factor for shorter overall survival (OS) in AML patients (p 0.031). Conclusion: SDF-1 dominant model had a higher risk to develop AML, and CXCR4 positive expression predicts poor prognosis in AML patients and it could represent a targeted therapy in AML. In addition, CXCR4 could be easily integrated into the initial routine diagnostic work up of AML.     

Clinical Significance of CD200 and CD56 Expression in Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) escape from immunosurveillance by immunosuppression. CD200 and CD56 expression represented an independent prognostic factor in many hematological malignancies but its importance in AML patients remains to be identified. Methods: CD200 and CD56 expression were assessed in the bone marrow blasts for Fifty-two (52) newly diagnosed AML by flowcytometry before start of therapy. Results: CD200+ expression was reported in 28.8% of patients while 17.3% of patients showed CD56+ expression. M4 FAB revealed high frequency of both CD200+ and CD56+ expression. The overall survival of CD200+ patients was 19.2% compared to 35.3% in CD200- (P= 0.049). On the other hand, CD56+ patients had the lowest complete remission rate (22.2% vs. 53.4%). In addition, CD56+ population had significant bad influence on overall survival than those of CD56- population (11.1 % vs. 35.5 %, P= 0.047). Conclusions: CD200 and CD56 positive expression by myeloblasts at diagnosis denote poor prognostic indicator and correlated with poor cytogenetic findings. CD200 could be used as therapeutic target in AML.     

急性髓系白血病患者HOX11基因的表达及意义

目的 探讨HOX11基因在急性髓系白血病(acute myeloid leukemia,AML)中的表达及对预后的影响,为个体化治疗提供依据.方法 应用多重巢式RT-PCR方法对73例初诊AML患者的融合基因进行检测,对HOX11基因表达阳性和阴性的患者进行标准治疗后的疗效进行分析.结果 在预后良好组、预后中等组及预后不良组AML患者的标准治疗中,HOX11基因阳性表达患者的第一疗程完全缓解率(complete remission rate)并不高于阴性表达的患者(P＞0.05),而复发或死亡率(relapse or mortality rate)与HOX11基因阴性表达的患者比较差异有统计学意义(P＜0.05).结论 HOX11基因的表达可能影响AML患者的预后.     

Effects of rhGM-CSF on Myeloid Clonogenic Cells in Acute Myelogenous Leukemia Patients

The effects of rhGM-CSF in vivo on the myeloid clonogenic cells present in 6 AML patients was evaluated. The relative number of clonogenic cells fell in 4 of the 6 patients. The effects of rhGM-CSF on the percentage of clonogenic cells in S phase and the sensitivity of clonogenic cells to cytosine arabinoside varied among the patients. These effects were not related to the effects of rhGM-CSF on the white blood cell count or on the proliferative rate of the leukemia cell population as a whole.     

TLR2mRNA和TLR4mRNA在蕈样肉芽肿皮损中的表达

目的:探讨Toll样受体2(TLR2) mRNA和Toll样受体4(TLR4) mRNA在蕈样肉芽肿皮损中的表达水平.方法:采用原位杂交技术检测蕈样肉芽肿皮损和正常皮肤中TLR2 mRNA和TLR4 mRNA的表达.结果:蕈样肉芽肿皮损中TLR2 mRNA和TLR4 mRNA主要表达于表皮基底上层,正常皮肤则表达于表皮的基底层.统计学分析表明TLR2 mRNA和TLR4 mRNA在正常皮肤和蕈样肉芽肿皮损处基底层和基底上层的表达均有显著性差异(P＜0.05),且TLR2 mRNA和TLR4 mRNA在蕈样肉芽肿皮损中的表达均高于在正常皮肤中的表达(P＜0.05).结论:TLR2 mRNA和TLR4 mRNA可能在蕈样肉芽肿的发生与发展过程中发挥重要作用.     

Prognostic Value of a CYP2B6 Gene Polymorphism in Patients with Acute Myeloid Leukemia

Background: The objectives of this study aimed to detect a CYP2B6 polymorphism in de novo cases of acutemyeloid leukemia patients and identify any role in disease progression and outcome. Materials and Methods:DNA was isolated from peripheral blood of 82 newly diagnosed acute myeloid leukemia cases and the CYP2B6G15631T gene polymorphism was assayed by PCR restriction fragment length polymorphism (PCR-RFLP).Results: The frequency of the GG genotype (wild type) was 48 (58.5%) and that of the mutant type T allele was 34(41.9%). GT genotype heterozygous variants were found in 28 (34%), and TT genotype homozygous variants in6 (7.3%) cases. We found no significant association between the CYP2B6 G15631T polymorphism and completeresponse (CR) (p-value=0.768), FAB classification (p-value=0.51), cytogenetic analysis (p-value=0.673), and overallsurvival (p-value=0.325). Also, there were no significant links with early toxic death (p-value=0.92) or progressionfreesurvival (PFS) (p-value=0.245). Conclusions: Our results suggest that the CYP2B6 polymorphism has norole in disease progression, therapeutic outcome, patient free survival, early toxic death and overall survival inacute myeloid leukemia patients.     

Targeting Apoptosis Pathways in Acute Myeloid Leukemia

The anti-apoptotic protein BCL2 is overexpressed in hematological malignancies, including acute myeloid leukemia (AML), favoring tumor survival and chemoresistance. BCL2 inhibits BIM and BAX, effector proteins necessary for the formation of pores in the mitochondrial outer membrane, and its inhibition primes cells for the release of cytochrome c and subsequent apoptosis. Such priming can be facilitated by venetoclax, an oral BCL2 inhibitor, therefore representing an ideal strategy to induce

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NOTCH-1 Gene Mutations Influence Survival in Acute Myeloid Leukemia Patients

Background: Although NOTCH-1 gene mutations were reported to contributes to leukemogenesis in lymphocytic leukemias, its role in acute myeloid leukemia (AML) remains unclear. Therefor; this study was designed to determine the prevalence and clinical impact of NOTCH-1 mutations in AML patients. Materials and Methods: In the current study, NOTCH-1 gene mutations were identified in Bone Marrow samples obtained from fifty primary AML patients before start of therapy using Sanger sequencing. Results: NOTCH-1 gene mutations were detected in 6 out of 50 AML cases (12%). The three mutations were (two mutations C7318A in the Pest domain exon 34); (another 2 in the Pest domain Del 7,344, ins C7349, G7356A and the last ones in the HD-N exon-26 (Del A4609). The clinical findings in the mutant AML (mu AML) patients did not significantly different as compared to the un mutated (unmut) AML patients.  There is significant association between CD7 aberrant expression and NOTCH-1 mutations. The complete remission was significantly higher in unmut AML cases as compared to mut AML ones (P=0.024). Multivariate (Age; Gender; Bone Marrow Blast cells; NOTCH-1 mutations) Cox regression analysis revealed that NOTCH-1 mutation is an independent risk factor for AML overall survival (P<0.001). The OS in unmut AML group (21.2 months) was significantly longer as compared to mut AML one (1.2 months) (P<0.001). Conclusion: Our data indicate that NOTCH-1 gene mutations were detected in 12% of AML patients. These mutations displayed bad clinical outcome on AML patients. Therapeutic targeting of NOTCH-1 could be a potentially effective approach to combat master oncogenic drivers in AML.     

Associations of IL-10 Gene Polymorphisms with Acute Myeloid Leukemia in Hunan,China

We investigated the possible association of interleukin-10 (IL-10) single nucleotide polymorphisms (SNPs)and susceptibility to acute myeloid leukemia (AML) in 115 patients and 137 healthy controls. Genetic analysisof IL-10 SNPs at -819 and -592 was carried out with the polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) approach. The IL-10 mRNA expression of AML patients and controls with differentgenotype was detected by real-time quantitative polymerase chain reaction (RT-PCR). Genetic analysis of IL-10revealed that the -819AA genotype frequencies and the -819A allele frequencies in the AML group were higherthan in the controls (59.1% vs 40.9%; 75.6% vs 63.9%, respectively); there were remarkable differences in-819T/C and -592A/C gene distribution (P<0.05) and the TA haploid frequencies were higher in the AML group(75.6% vs 63.9%, P<0.05). IL-10 mRNA expression in incipient AML patients was obvious higher than the nontumorgroup and the remission group (7.78×10-3 vs 2.43×10-3, 3.64×10-3, P<0.05).The study suggested that thehaploid TA and genotype TA/TA may be associated with AML in Han people in Hunan province.The IL-10 SNPsat -819 and -592 sites were associated with AML and may affect IL-10 mRNA expression in AML patients.     

Role of the MDM2 Promoter Polymorphism (-309T>G) in Acute Myeloid Leukemia Development

Background: The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator ofTp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpressionthereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate thepossible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. Materials andMethods: A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for theMDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCRmethod. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantifiedMDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypesand the MDM2 expression were correlated with disease free survival (DFS) rates among patients who haveachieved complete remission (CR) after first induction chemotherapy. Results: MDM2-309T>G polymorphismwas significantly associated with AML development (p<0.0001). The presence of either GG genotype or G alleleat MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p= 0.003) increasedAML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates(16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+vecases (p=0.0036). Conclusions: The MDM2-309T>G polymorphism might be involved in AML development andalso serve as a good prognostic indicator.     

Impact of FLT3 Receptor (CD135) Detection by Flow Cytometry on Clinical Outcome of Adult Acute Myeloid Leukemia Patients

Background

The significance of FMS-like tyrosine kinase 3 (FLT3)-ITD mutation in acute myeloid leukemia (AML) prognosis has been well established. The aims of this study were to investigate the prognostic impact of the FLT3 protein (CD135) expression and its association with FLT3-ITD mutation, and to identify its role in minimal residual disease.

CXC Chemokine Receptor 4 Expression,CXC Chemokine Receptor 4 Activation,and Wild-Type Nucleophosmin Are Independently Associated With Unfavorable Prognosis in Patients With Acute Myeloid Leukemia

BackgroundCXC chemokine receptor 4 (CXCR4) is activated by phosphorylation and essential for migration of hematopoietic precursors to bone marrow. CXCR4 overexpression predicts unfavorable prognosis in patients with acute myeloid leukemia (AML). Nucleophosmin (NPM1) mutation is the most frequent genetic abnormality in patients with AML and predicts a favorable prognosis. In vitro studies have suggested that mutant nucleophosmin (NPM) decreases CXCR4-mediated chemotaxis by downregulating CXCR4, thereby linking the NPM and CXCR4 pathways.Patients and MethodsIn a group of 117 untreated adults with AML, we used immunohistochemistry to assess bone marrow specimens for CXCR4 and phosphorylated CXCR4 (pCXCR4) expression. All cases also were analyzed for NPM1 mutations using polymerase chain reaction–based methods.ResultsCXCR4 expression was detected in 75 patients (64%), and pCXCR4 expression was detected in 31 patients (26%). NPM1 mutations were detected in 63 patients (54%). NPM1 mutations did not correlate with CXCR4 (P = .212) or pCXCR4 (P = .355) expression. The median 5-year overall survival was 27% (95% confidence interval, 19-36), with a median follow-up of 8 months (95% confidence interval, 6-15). In a multivariate Cox proportional hazards model, reduced overall and progression-free survival rates were associated with a history of antecedent hematologic disorder, failure to achieve complete remission, thrombocytopenia, unfavorable cytogenetics, CXCR4 expression, and wild-type NPM1. pCXCR4 expression was independently associated with shorter progression-free survival.ConclusionsThere is no correlation between NPM1 mutations and CXCR4 or pCXCR4 expression, suggesting that the CXCR4 and NPM pathways act independently in adult AML.     

Tracing Leukemia Stem Cells and Their Influence on Clinical Course of Adult Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) evolves from neoplastic transformation of stem cell disease termed “leukemia stem cells” (LSCs). An unsatisfactory response to AML therapy is determined by the presence of minimal residual disease (MRD). The predominance of LSCs might anticipate sustained MRD results. The present study aimed to demonstrate the effect of LSCs on MRD at induction days 14 and 28 on overall survival (OS) and disease-free survival (DFS) and to compare LSC expression with MRD status.Patients and MethodsA total of 84 patients with de novo adult AML underwent testing using LSC panels for CD38/CD123/CD34/CD45 and CD90/CD133/CD45/CD33 and different regular MRD panels.ResultsAt day 14 after induction, the high expression of CD123 and CD133 had adverse effects on both OS and DFS (P = .004 and P ≤ .001 and P ≤ .001 and P ≤ .001, respectively). Greater expression of CD34⁺/CD38⁻/CD123⁺ resulted in unfavorable OS and DFS (P ≤ .001 for both). Both CD34⁺/CD38⁻/CD123⁺ and CD34⁻/CD38⁺/CD123⁺ expression at day 14 after induction had an adverse effect on DFS only (P < .001 and P = .029, respectively). On multivariate analysis, CD133 expression and MRD status were independent prognostic parameters (hazard ratio [HR], 2.3; 95% confidence interval [CI], 1.2-4.4; P = .015; and HR, 2.9; 95% CI, 1.0-7.9; P = .041). At day 28 after induction, MRD and increased CD123⁺/CD34⁻, CD34⁺/CD38⁻/CD123⁺, CD133⁺/CD33⁻ expression were associated with inferior OS (P = .016, P = .0035, P = .0.002, and P = .002, respectively). MRD and high expression of CD34⁺CD123⁺, CD133⁺/CD33⁻, CD34⁺/CD38⁻/CD123⁺ were associated with inferior DFS (P < .001, P = .002, P < .001, P < .001, respectively). On multivariate analysis, only CD133⁺/CD33⁻ expression was the independent prognostic factor (HR, 3.1; 95% CI, 1.5-6.7; P = .003).ConclusionsEstimation of LSC expression is a sensitive indicator of the response to therapy in adult patients with AML and might be a better prognosticator than the findings from regular MRD panels.     

Kinetic and Immunophenotypic Heterogeneity in Acute Myeloid Leukemia Subpopulations

Bone marrow samples of 27 cases of acute myeloid leukemia were studied by double staining with 3H-Thymidine and monoclonal antibodies (APAAP method). 3H autoradiography labels the nucleus of S-phase cells and immunocytochemistry (APAAP) detects cytoplasmic antigens. Different myeloid subsets were characterized with the monoclonal antibodies My7 (CD13), My9 (CD33), My4 (CD14), IOM11c (CD11c), Ber Mac3 and anti-HLA-DR. We have shown that there are different myeloid subsets with different proliferative patterns. The most notable finding was that in M4 acute leukemia only myeloid subsets were in the S-phase, while in M5 acute leukemia the highest percentage of proliferating cells was in the monocytic subsets.