Effect of DNMT3A R882H Hot Spot Mutations on DDX43 Promoter Methylation in Acute Myeloid Leukemia.

Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in DNA methyl transferase (DNMT) enzymes, disabling them to methylate target genes in a proper way. In this case-control study, we investigated the association between R882H mutation in DNMT3A gene and DDX43 gene methylation in patients with AML. 47 AML patients and 6 controls were included in this study. After DNA extraction, amplification refractory mutation system (ARMS)-PCR was used to evaluate R882H mutations in DNMT3A gene. The high-resolution melting (HRM) method was used to determine the methylation changes of the DDX43 gene promoter. R882H mutation was only found in 10.6% (5 out of 47) of AML patients. The frequency of DDX43 gene methylation was significantly higher in patients without R882H mutations compared to patients with R882H mutations (P < 0.05). The DNMT3A R882H mutation is typically present in a minority of AML patients. Nevertheless, this mutation is associated with a reduced frequency of methylation in the DDX43 promoter region.

Evaluation of bacterial safety approaches of platelet blood concentrates: bacterial screening and pathogen reduction.

This mini-review analyzed two approaches to screening bacterial contamination and utilizing pathogen reduction technology (PRT) for Platelet concentrates (PCs). While the culture-based method is still considered the gold standard for detecting bacterial contamination in PCs, efforts in the past two decades to minimize transfusion-transmitted bacterial infections (TTBIs) have been insufficient to eliminate this infectious threat. PRTs have emerged as a crucial tool to enhance safety and mitigate these risks. The evidence suggests that the screening strategy for bacterial contamination is more successful in ensuring PC quality, decreasing the necessity for frequent transfusions, and improving resistance to platelet transfusion. Alternatively, the PRT approach is superior regarding PC safety. However, both methods are equally effective in managing bleeding. In conclusion, PRT can become a more prevalent means of safety for PCs compared to culture-based approaches and will soon comprehensively surpass culture-based bacterial contamination detection methods.

Complications in patients with transfusion dependent thalassemia: A descriptive cross-sectional study.

Background and Aims: One of the most common hemoglobinopathies globally related to blood transfusion and iron overload in the body is thalassemia syndrome. Increasing ferritin levels can cause severe damage to the patient's body organs. This study aims to evaluate the complications of iron overload on vital body organs in patients with transfusion-dependent beta-thalassemia. Methods: This descriptive cross-sectional study was performed in Iran University of Medical Sciences Hospitals on patients with a beta-thalassemia major with frequent blood transfusions. To evaluate the effect of iron overload on vital body organs, hematologic and blood analysis, echocardiography with measurement of pulmonary artery pressure (PAP) and ejection fraction (EF) tests, bone densitometry, and audiometric tests were performed for all patients. Results: Of the 1010 patients participating in this study, 497 (49%) were males, 513 were (51%) females aged 5-74 years, and the majority of participants (85%) were over 20 years old. This study demonstrated that increasing ferritin levels had no notable correlation with sex, cholesterol, low-density lipoprotein, parathyroid hormone, T4, and aspartate aminotransferase. However, elevating ferritin levels had significant correlations with increasing triglyceride, phosphorus, thyroid stimulating hormone, alkaline phosphatase, alanine transaminase, and PAP levels, age, hearing disorders, splenectomy, osteoporosis, and decreasing high-density lipoprotein, body mass index, calcium, and EF levels. Conclusion: Improvement in beta-thalassemia patients' survival and quality of life can be due to multidisciplinary care in a comprehensive unit through regular follow-up and early complication detection.

Studying the potential of upregulated PTGS2 and VEGF-C besides hyper-methylation of PTGS2 promoter as biomarkers of Acute myeloid leukemia.

Hereby, we aimed to investigate the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and Vascular Endothelial Factor-C (VEGF-C) besides the methylation of PTGS2 in AML patients. VEGF-C and PTGS2 expression analysis were evaluated in newly diagnosed AML patients and healthy controls by quantitative Reverse Transcriptase PCR method. Also, PTGS2 methylation status was evaluated by Methylation-Sensitive High-Resolution Melting Curve Analysis (MS-HRM). While 34% of patients were female, the mean age of the patients was 43.41 ± 17.60 years suffering mostly from M4 (48.21%) type of AML. Although methylation level between patients and controls was not significantly different, none of the normal controls showed methylation in the PTGS2 promoter. PTGS2 and VEGF-C levels were elevated in AML cases and correlated with WBC, Platelet, and Hemoglobin levels. The survival of patients with overexpressed VEGF-C and PTGS2 was poorer than others. It can be concluded that PTGS2 and especially VEGF-C expression but not PTGS2 methylation can be considered as diagnostic biomarkers for AML.

Use of antioxidant nanoparticles to reduce oxidative stress in blood storage.

Oxidative damage by free radicals has a negative effect on blood quality during storage. Antioxidant nanoparticles can prevent oxidative stress. We use SOD-CAT-Alb-PEG-PLGA- nanoparticles to reduce the effects of oxidative stress in blood storage. Electrospray was employed to prepare nanoparticles. Nanoparticles entered the test bags and were kept for 35 days from the time of donation under standard conditions. On target days, experiments were performed on the samples taken. The examination included blood smear, red blood cells count, hemoglobin, hematocrit, K, Fe, glutathione peroxidase, glutathion reductase, glucose-6-phosphate dehydrogenase, prooxidant-antioxidant balance, malondialdehyde, and flow cytometric assay for phosphatidylserine. The repeated measures analysis was performed on samples every week. Morphological changes were less in the test group compared to the control. The quantitative hemolysis profile test showed significant changes in the test and control groups (p < 0.05) in consecutive weeks except for K and Fe. Oxidative stress parameters too showed a significant change during the target days of the examination (p < 0.05). Also, the phosphatidylserine expression was increased in control groups more than test in consecutive weeks (p < 0.05). It seems that the use of antioxidant nanoparticles improves the quality of stored red blood cells and can prevent posttransfusion complications and blood loss by reducing oxidative stress.

Polymorphisms within methotrexate pathway genes: Relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia.

OBJECTIVES: The current study aimed to investigate the relationship of genetic polymorphism and plasma methotrexate (MTX) levels, toxicity experience and event free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: The study included 74 ALL patients. Polymerase chain reaction and genotyping of methylene tetrahydrofolate reductase (MTHFR) rs1801133, MTHFR rs1801131, ATP-binding cassette superfamily B1 (ABCB1) rs1045642, ATP-binding cassette superfamily G2 (ABCG2) rs2231142 and solute carrier 19A1 (SLC19A1) rs1051266 genetic variations were performed. The plasma MTX levels were investigated at 48 hr after the first dose of MTX infusion. RESULTS: MTHFR rs1801133 TT genotype, ABCBa1 rs1045642 CT genotype and ABCG2 rs2231142 CA genotype revealed a statistically significant association with the MTX plasma levels (P<0.01, P<0.05, P<0.05, respectively). The MTHFR rs1801133 TT genotype had a statistically significant association with hematopoietic toxicity (P<0.01) and interventions (P<0.05). The MTHFR rs1801131 AC genotype was related to the decreased hepatic toxicity (P<0.05). The SLC19A1 rs 1051266 GA genotype was related to the increased hepatic toxicity (P<0.05). Only the ABCB1 rs1045642 CT and TT genotypes had a statistically significant correlation with EFS (P<0.05, P<0.05, respectively). CONCLUSION: Our findings showed that genetic polymorphism could be associated with plasma MTX levels, toxicity experienced and EFS in Iranian pediatric ALL.

Effects of Reduced Mir-24 Expression on Plasma Methotrexate Levels, Therapy-Related Toxicities, and Patient Outcomes in Pediatric Acute Lymphoblastic Leukemia.

BACKGROUND: The current study aims to investigate the relationship of miR-24 expression with plasma methotrexate (MTX) levels, therapy-related toxicities, and event-free survival (EFS) in Iranian pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: The study included 74 ALL patients in consolidation phase and 41 healthy children. RNA was extracted from plasma, polyadenylated, and reverse transcribed. miR-24 expression was determined by quantitative polymerase chain reaction (qPCR). Plasma MTX concentrations were measured by high performance liquid chromatography (HPLC) 48 h after high-dose methotrexate (HD-MTX) injection. The diagnosis of ALL was further subclassified as B-ALL or T-ALL via flow cytometry. RESULTS: miR-24 expression was less in pediatric ALL patients than in the control group (p = 0.0038). Furthermore, downregulation of miR-24 was correlated with intermediate- to high-grade HD-MTX therapy toxicities (p = 0.025). Nevertheless, no statistically significant associations were seen between miR-24 levels and plasma MTX levels 48 h after HD-MTX administration (p > 0.05) or EFS in pediatric ALL patients (p > 0.05). CONCLUSION: miR-24 expression may contribute to interindividual variability in response to intermediate- to highgrade HD-MTX therapy toxicities under Berlin Frankfurt Munster (BFM) treatment.

Pattern of ABCC Transporter Gene Expression in Pediatric Patients with Relapsed Acute Lymphoblastic Leukemia.

BACKGROUND: Abnormal expression of ABCC transporter genes has been associated with treatment failure in pediatric patients with acute lymphoblastic leukemia (ALL). The aim of this study was to evaluate the expression pattern of ABCC1-6 and ABCC10 genes in Iranian pediatric patients with ALL relapse and determine the potential predictive value of determining ALL relapse from ABCC expression. METHODS: Patients with ALL were divided into two separate groups, either the case group with relapsed ALL or the control group in which ALL patients have been in progression-free survival for at least 3 years A total of thirty-nine participants (23 with relapsed ALL; 16 controls) were enrolled over 26 months. To determine the levels of ABCC1-6 and ABCC10 transporter gene expression RT-PCR was used. Cumulative doses of the chemotherapy drugs, VCR, DNR and L-ASP, were calculated for each patient. RESULTS: Our findings showed elevated expression of ABCC2-6 and decreased expression of ABCC1 and ABCC10 to be associated with an increased risk of ALL relapse. The mean-fold expression of ABCC2 was significantly increased in the ALL relapse group. Additionally, the expression pattern of the ABCC transporter genes was associated with high doses of three chemotherapy drugs, VCR, DNR and L-ASP. CONCLUSION: Evaluating the expression pattern of ABCC transporter genes may be a potential biomarker for predicting the occurrence of ALL relapse in Iranian pediatric patients and improve cancer prognosis.

ATP-Binding Cassette transporters' gene expression in pediatric patients with acute leukemia; a comprehensive analysis of published reports through PubMed search engine.

Multidrug resistance based on ABC transporters' gene expression is one of the most important health challenges through chemotherapy of patients. This resistance can cause relapse or treatment failure. The goal of this conducted study was to evaluate the results of published reports which considered ABC transporters' gene expression in pediatric patients with acute leukemia. PubMed as a free search engine was chosen. The following Mesh terms were used as: "ATP-binding cassette transporters" OR "ABC-transporters*" AND "gene expression*" AND "leukemia" OR "ALL" OR "AML" OR "acute leukemia*". Age was set as an additional filter with the age range of birth to 18 years old. Initial screening was performed according to inclusion and exclusion criteria and the quality of the selected papers was assessed. Papers categorized into three sections as: pediatric patients with ALL (6 papers from 1998-2015); pediatric patients with AML (3 papers from 1992-2011) and pediatric patients with ALL and AML (7 papers from 1992-2014). Totally 1118 patients enrolled in the searched studies (ALL and AML: 488; ALL: 405; AML: 225). The common method for evaluating gene expression of ABC transporters was RT-PCR. More than 50% of the papers showed the influence of ABC transporters' gene expression on prognosis and treatment failures of patients. Despite controversial results, the gathered information in the current report serves as a comprehensive referential resource, which can be beneficial for future planning around this title, especially in developing countries.

Frequency of three common mutations of CARD15/NOD2 gene in Iranian IBD patients.

BACKGROUND: The CARD15/NOD2 gene, located on the pericentromeric region of chromosome 16 (IBD1) has been reported to have an association with IBD, especially Crohn's disease. Three common mutations of CARD15 are variably associated with Crohn's disease in different ethnic groups. We evaluated the frequency of these mutations (R702W, G908R and 1007fsinsC) in Iranian IBD patients and compared it with the healthy control population. METHODS: One hundred patients with ulcerative colitis, 40 patients with Crohn's disease, and 100 sex- and age-matched controls were enrolled from a tertiary center during a one-year period (2005-2006). The three mutations were assessed in DNA of leukocytes by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS: The frequency of R702W mutation was significantly higher in Iranian patients with Crohn's disease (p< 0.001; OR 19.21; 95% CI 4.23-87.32) compared to healthy controls. No association was observed between the other mutations and Crohn's disease and none of these mutations was associated with ulcerative colitis. CONCLUSION: The R702W mutation of CARD15 gene was associated with Crohn's disease in the Iranian population.

Analysis of HLA-G gene expression in B-lymphocytes from chronic lymphocytic leukemia patients.

BACKGROUND: The human leukocyte antigen G (HLA-G) molecule exhibits limited tissue distribution, low polymorphism and alternative splicings that generate seven HLA-G isoforms. HLA-G exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells that may favor their escape from anti-tumor immune responses. This study it is an effort to clarify the presence of HLA-G in B-cell chronic lymphocytic leukemia (B-CLL) patients. METHODS: HLA-G mRNA expression was studied in a pilot study in circulating B-CLL and also healthy controls by reverse transcription (RT)-PCR using a set of pan-HLA-G primers. RESULTS: RT-PCR was performed on B-cells from 74 B-CLL patients and 12 healthy controls. The data showed HLA-G gene expression in 20% of the B-CLL patients. No expression of HLA-G could be detected in the healthy control group. CONCLUSION: These data suggest that HLA-G is expressed at the gene level in B cells from B-CLL patients but not in B cells from healthy controls. Further study is required to clarify the role of HLA-G as a regulatory factor that could affect immune response in B-CLL patients.

Reconstitution of the T-cell repertoire following treatment with alemtuzumab (anti-CD52 monoclonal antibody) in patients with B-cell chronic lymphocytic leukaemia.

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 (P < 0.01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells.

Fibromodulin, an extracellular matrix protein: characterization of its unique gene and protein expression in B-cell chronic lymphocytic leukemia and mantle cell lymphoma.

Fibromodulin is an extracellular matrix protein normally produced by collagen-rich tissues; the fibromodulin gene has been found to be the most overexpressed gene in B-cell chronic lymphocytic leukemia. In this study, fibromodulin was expressed at the gene level (reverse transcription-polymerase chain reaction [RT-PCR]) in all patients with B-CLL (n = 75) and in most (5 of 7) patients with mantle cell lymphoma (MCL). No mutations in the fibromodulin gene were detected. Fibromodulin was also detected at the protein level in the cytoplasm of the B-CLL cells and in the supernatant after in vitro cultivation, but not at the cell surface. Fibromodulin was not found in patients with T-cell chronic lymphocytic leukemia (T-CLL), B-cell prolymphocytic leukemia (B-PLL), T-cell prolymphocytic leukemia (T-PLL), hairy cell leukemia, follicular lymphoma, lymphoplasmacytic lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), or chronic myelogenous leukemia (CML) or in 36 hematologic cell lines. Normal blood mononuclear cells (T and B lymphocytes, monocytes), tonsil B cells, and granulocytes did not express fibromodulin. Activation (phorbol 12-myristate 13-acetate [PMA]/ionomycin) of normal T and B lymphocytes induced weak fibromodulin gene expression, but not to the extent seen in freshly isolated B-CLL cells. The reason for the exclusive ectopic expression of fibromodulin in B-CLL and MCL is unknown. However, its unique protein expression makes it likely that fibromodulin is involved in the pathobiology of B-CLL and MCL.

T-cell receptor BV gene usage in colorectal carcinoma patients immunised with recombinant Ep-CAM protein or anti-idiotypic antibody.

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-gamma production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-gamma T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response.

Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia.

T-cell receptor-B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell-specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell-associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.