Abnormal frequency of the memory B cell subsets and plasmablasts in patients with congenital severe hemophilia A: correlation with "Inhibitor" formation.

BACKGROUND: Development of antibodies against infused Factor VIII (FVIII) or "inhibitors" represents a major challenge following FVIII replacement therapy in patients with hemophilia A (HA). Recent studies have shown that certain cellular compartments of the immune system contribute to the production of such antibodies. Herein, we determined the frequency of class-switched CD19+IgD-CD27+/non-class-switched CD19+IgD+CD27+ memory B cell subsets and CD19+CD27hiCD38hi plasmablasts in patients with severe HA and their association with the development of inhibitors in these patients. METHODS: This cross-sectional case-control study enrolled 32 patients with severe HA, including 8 with and 24 without inhibitors, and 24 healthy individuals. The frequencies of the memory B cell subsets and plasmablasts were determined using flow cytometry. RESULTS: The frequency of CD19+IgD+CD27+ non-class-switched memory B cells was significantly lower in patients with HA (including both patients with and without inhibitors) than in healthy controls. The percentages of both CD19+IgD-CD27+ class-switched and CD19+IgD+CD27+ non-class-switched memory B cells did not differ significantly between patients with and without inhibitors. HA patients with inhibitors had significantly higher proportions of CD19+CD27hiCD38hi plasmablasts than the control group as well as the inhibitor (-) ones. No significant correlation was observed between the inhibitor levels with the percentages of memory B cell subsets and plasmablasts. CONCLUSION: This study is the first to demonstrate a dysregulated proportion of CD19+IgD+CD27+ non-class-switched memory B cells and CD19+CD27hiCD38hi plasmablasts in patients with severe HA. Therefore, strategies targeting memory B-cell/plasmablast differentiation may have promising outcomes in the management of inhibitor formation in patients with severe HA.

Altered frequency of FOXP3+ regulatory T cells is associated with development of inhibitors in patients with severe hemophilia A.

INTRODUCTION: The development of anti-factor VIII (FVIII) antibodies or "inhibitors" is a major complication following FVIII replacement therapy in patients with severe hemophilia A (HA), rendering the treatment inefficient. Data on the role of regulatory T cells (Tregs) in inhibitor formation in these patients are rare. Herein, we aimed to investigate whether a difference in the FOXP3+ Tregs is linked to the formation of the inhibitors in severe HA patients. METHODS: In this cross-sectional study, 32 patients with severe HA (8 patients with inhibitors and 24 without inhibitors) and 24 healthy controls were enrolled. The frequency of FOXP3+ Tregs was determined using multicolor flow cytometry method. RESULTS: Our results showed that the median level of CD4+ CD25+ FOXP3+ Tregs did not significantly differ between HA patients and healthy controls and between HA patients with and without inhibitors (P > 0.05). However, patients with inhibitors had significantly lower amounts of CD4+ CD25- FOXP3+ Tregs compared to those without inhibitors as well as healthy controls (*P = 0.012 and *P = 0.004, respectively). The frequency of CD4+ CD25+ T cells was significantly higher in HA patients who developed inhibitors compared to the inhibitor-negative ones whereas they were lower in inhibitor-negative patients compared to the healthy controls (*P = 0.013 and *P = *0.029, respectively). The percentages of CD4+ CD25+ T cells were positively correlated with the levels of inhibitors in HA patients (r = 0.45, *P = 0.021). CONCLUSION: Our data demonstrated for the first time that the CD4+ CD25- FOXP3+ Tregs might be implicated in the prevention of inhibitor formation in severe HA patients.

Selenium Supplementation Alter the Frequency of Th17 but not Th1/Th2 Lymphocytes in Diffuse Large B Cell Lymphoma Patients.

BACKGROUND: Selenium (Se) is a micronutrient, which has recently been proven to have a positive effect on the immune system of cancer patients, but the underlying mechanism is not clearly defined. In this randomized controlled trial, we evaluated the effect of three-month Se supplementation on the profile of CD4+ T-helper subsets including IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 cells in sixteen diffuse large B cell lymphoma (DLBCL) patients at stable remission phase who consumed Se (Se+) compared to the fourteen control patients who did not receive Se (Se-). METHODS: The frequency of IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 lymphocytes was determined using a four-color flow cytometry method. RESULTS: The results revealed that three-month Se supplementation significantly decreased the proportion of CD4+IL-17+ Th17 lymphocytes but not IFN-γ+/IL-4- Th1 and IFN-γ-/IL-4+ Th2 subtypes in DLBCL patients at stable remission. Change in the percentage of IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 cells did not significantly differ between Se+ and Se- groups. No positive correlation was observed between changes in different Th subpopulations in both Se+ and Se- groups. CONCLUSIONS: Taken together, three-month Se supplementation can reduce the proportion of CD4+IL-17+ Th17 cells in DLBCL patients at stable remission phase. Larger population and longer follow-up of patients is necessary to specify the clinical significance of Se supplementation on the popularity of T-helper cells in DLBCL patients.

Atopy manifestations in pediatric patients with acute lymphoblastic leukemia: correlation assessment with interleukin-4 (IL-4) and IgE level.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in the age range of under 15 years old and accounts for 25-30% of all childhood cancers. Although conventional chemotherapy regimens are used to improve the overall survival rate, it has been associated with some complications, amongst which allergic manifestations with unknown mechanisms are more common. METHODS: Our study compared serum IgE and IL-4 concentration, as a hallmark of allergic responses in pediatric ALL patients before and after 6 months of intensive (high-dose) chemotherapy, to show whether changes in the level of these markers may be associated with atopy. Serum level of IL-4 and IgE was measured using enzyme-linked immunosorbent assay (ELISA) method. RESULTS: The results showed that the level of IgE and IL-4 increased following chemotherapy in both ALL patients with and without atopy. In addition, post-chemotherapy treatment IgE and IL-4 levels were significantly elevated in patients with atopy compared to those without it. The difference between baseline and post-chemotherapy level of IgE and IL-4 was significantly higher in patients with atopy compared to those without it. CONCLUSIONS: To the best of our knowledge, this is the first study that showed a connection between post-chemotherapy allergic manifestations in pediatric ALL patients and IL-4 and IgE level. Flow cytometry analysis of the T-helper 2 (Th2) lymphocytes and other allergy-related T cell subsets like Tc2 and Th9 as well as the study of the genetic variations in atopy-related genes like IL-4/IL-4R, IL-5, IL-9, IL-13, and high affinity FcεRI IgE receptor and also HLA genes is necessary to clearly define the underlying mechanism responsible for post-chemotherapy hypersensitivity reaction in pediatric ALL patients.

The prognostic significance of immune checkpoint receptor expression in patients with lymphoma: Association with disease status and clinical outcomes.

INTRODUCTION: Little is known about the expression of immune checkpoint receptors in the peripheral blood of lymphoma patients. Herein, we assessed the expression of inhibitory checkpoint receptors, including CTLA-4, PD-1/PDL-1, LAG-3, and TIM-3 in the peripheral blood of lymphoma patients and its correlation with the clinical outcomes of patients. Therefore, 47 classical Hodgkin lymphoma (cHL), 48 non-Hodgkin lymphoma patients with diffuse large B-cell lymphoma (DLBCL) subtype, and 30 healthy controls were recruited. METHODS: The expression of inhibitory receptors was evaluated using SYBR Green real-time PCR method. RESULTS: CTLA-4, LAG-3, and TIM-3 genes were significantly upregulated in both cHL and DLBCL patients compared to the healthy controls. In addition, the level of these molecules was differentially expressed in cHL and DLBCL patients at different disease phases compared to the healthy controls. The CTLA-4 gene was highly expressed in newly diagnosed (ND) cHL patients compared to the relapsed ones. Relapsed DLBCL patients had significantly increased LAG-3 expression compared to patients at remission, as well as ND patients. Regarding cHL patients, high CTLA-4 expression was correlated with low lactate dehydrogenase level and better performance status, whereas the level of LAG-3 was significantly elevated in patients with poor performance status. Lower initial PD-1 expression was associated with improved disease-free survival in cHL patients. CONCLUSIONS: Inhibitory immune checkpoint receptors are aberrantly expressed in the peripheral blood of cHL and DLBCL patients in which high LAG-3 in DLBCL patients and PD-1/LAG-3 in cHL patients are associated with relapse occurrence and worse prognosis, respectively.

Higher Peripheral Blood IFN-γ-/IL-4+ Th2 Lymphocytes Are Associated with Lower Rate of Relapse in Patients with Lymphoma.

BACKGROUND: The role of T-helper lymphocytes especially T helper 2 (Th2) subsets in lymphoid malignancies is debatable and unknown. METHODS: Herein, we evaluated the polarization of the IFN-γ+/IL-4- Th1 and IFN-γ-/IL-4+ Th2 lymphocytes in 95 lymphoma patients including 47 classical Hodgkin's lymphoma (cHL) and 48 diffuse large B cell lymphoma patients (DLBCL) at different disease phases and its correlation with the clinical outcomes of patients using flow cytometry method. RESULTS: The proportion of IFN-γ+/IL-4- Th1 lymphocytes was significantly higher in cHL patients at remission compared to the newly diagnosed ones. Both cHL and DLBCL patients at remission phase had significantly more IFN-γ-/IL-4+ Th2 lymphocytes than those patients at relapse/refractory phase as well as newly diagnosed ones. Despite having higher frequency of IFN-γ+/IL-4- Th1 lymphocytes, the mean fluorescent intensity (MFI) of IFN-γ was lower in relapsed cHL patients, in those with high-risk IPI score, performance status (PS) ≥2 and B symptom-positive groups compared to their corresponding counterparts in newly diagnosed patients. CONCLUSION: Taken together, higher peripheral blood IFN-γ-/IL-4+ Th2 lymphocytes might be associated with a favorable prognosis like lower rate of relapse in lymphoma patients.

Study of the Serum Immunoglobulin and Cell-Mediated Immunity in Patients with Congenital Severe Hemophilia.

BACKGROUND: It has been shown that a close relationship exists between the immune system and coagulation cascade. Hemophilia A is an X-linked, recessive bleeding disorder caused by deficiency of functional plasma clotting factor VIII that is classically treated with factor VIII replacement therapy. Despite this, some patients produce inhibitors or antibodies against epitopes of infused factor VIII, indicating the activation of the adaptive immune system. The aim of this study was to evaluate the change in the T cell frequency and serum immunoglobulin level in patients with congenital hemophilia A, especially those who produce inhibitors against factor VIII. METHODS: This is a cross-sectional, case-control study on congenital hemophilia A patients with severe factor VIII deficiency. Twenty-eight hemophilia A male patients were randomly selected along with twenty age-matched healthy males, as the control group. Serum immunoglobulin concentration was measured by nephelometry (for IgG, IgA and IgM) and enzyme-linked immunosorbent assay (ELISA) (for IgE) and the frequency of CD4+ and CD8+ T cells were calculated using a flow cytometry method. RESULTS: Serum IgG was significantly higher in hemophilic patients compared to controls (14.35 ± 3.60, vs. 12.4 ± 1.72, p = 0.014). Among IgG subtypes, the IgG1 antibody was significantly higher in hemophilia patients than control group (p < 0.001). The frequency of CD4+ as well as CD8+ T cells did not significantly differ between patients and control group (p > 0.05). There was no significant difference between patients with and without inhibitors re-garding serum immunoglobulin level, different IgG subtypes, the frequency of CD4+ and CD8+ T cells as well as CD4/CD8 ratio (p > 0.05). CONCLUSIONS: Patients with hemophilia A have high levels of serum immunoglobulin especially IgG1. Therefore, further larger studies along with close observation and evaluation of the presence of serum inhibitor is recommended every 3 months.

Expression of the immune checkpoint receptors CTLA-4, LAG-3, and TIM-3 in ß-thalassemia major patients: correlation with alloantibody production and regulatory T cells (Tregs) phenotype.

Alloimmunization is a serious complication in ß-thalassemia major patients as a result of repeated blood transfusion. The immune checkpoint receptors play an important role in regulating immune system homeostasis and the function of the immune cells. This study aimed to evaluate the expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) immune checkpoint molecules in ß-thalassemia major patients with and without alloantibody. For this purpose, 68 ß-thalassemia major patients with (34 patients) and without (34 patients) alloantibody as well as 20 healthy controls were enrolled. The expression of these genes was evaluated in different groups of patients by SYBR Green real-time PCR method. Our results showed that the mean expression of LAG-3 was significantly increased in thalassemia patients compared to the control group (*P < 0.001). However, there was no significant difference in expression of the CTLA-4 and TIM-3 as well as LAG-3 genes between patients with and without alloantibody (P > 0.05). A positive correlation was observed between the level of LAG-3 expression with markers associated with Treg function including FOXP3 and GDF-15 genes in ß-thalassemia major patients. Taken together, the LAG-3 molecule might have a more prominent role in the abnormality of the immune system in thalassemia patients especially the function of regulatory T cells (Tregs), prior to the CTLA-4 and TIM-3 genes.

The impact of selenium on regulatory T cell frequency and immune checkpoint receptor expression in patients with diffuse large B cell lymphoma (DLBCL).

For many decades, selenium (Se) has been known as a potential anti-cancer agent that can also improve the function of immune cells in a variety of solid tumors. However, there is no report on the role of Se on CD4+ T cell subsets like CD4+CD25+FOXP3+ regulatory T cells (Tregs) in lymphoma patients. In this randomized clinical trial, we investigated the effect of 3-month Se consumption on the frequency of CD4+CD25+FOXP3+ Tregs and the expression of immune checkpoint receptors in thirty-two non-Hodgkin lymphoma (NHL) patients (16 patients with Se (Se+) and 16 without Se (Se-) consumption) with diffuse large B-cell lymphoma (DLBCL) subtype at stable remission. The change in the frequency of Tregs and expression of immune checkpoint receptors including CTLA-4, LAG-3, TIM-3, and PD-L1 genes were evaluated after 3 months in both groups using flow cytometry and SYBR Green Real-time PCR method, respectively. The results showed that the frequency of CD4+CD25+FOXP3+ Tregs and expression of immune checkpoint receptors did not significantly change after 3-month Se consumption in DLBCL patients. However, alteration in the frequency of CD4+CD25-FOXP3+ Treg subsets was positively correlated with change in CTLA-4, LAG-3, and TIM-3 expression in the Se+ group. Three-month Se supplementation did not prevent relapse in Se+ group. Taken together, Se supplementation alone did not affect the frequency of CD4+CD25+FOXP3+ Tregs, expression of checkpoint receptors, and prevention of relapse in DLBCL patients at stable remission phase but might influence the functional properties of other Treg subsets like CD4+CD25-FOXP3+ Tregs.

Association Between Cytotoxic T-Lymphocyte Antigen 4 Gene Polymorphisms and Torque Teno Virus Infection After Hematopoietic Stem Cell Transplantation.

OBJECTIVES: An association between costimulatory molecule gene polymorphisms and viral infection after hematopoietic stem cell transplantation may be related to clinical outcomes, especially acute graft-versus-host disease. Cytotoxic T-lymphocyte antigen 4 has been suggested as a crucial negative regulator of the immune system. In this study, our objective was to investigate the association between cytotoxic T-lymphocyte antigen-4 gene polymorphisms (including -1722 T/C, -1661 A/G, -318 C/T, and +49 A/G) and torque teno virus infection after hematopoietic stem cell transplantation in patients with and without acute graft-versus-host disease. MATERIALS AND METHODS: Our study included 71 recipients. We evaluated cytotoxic T-lymphocyte antigen 4 gene polymorphisms using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Our results showed that the GG genotype of the cytotoxic T-lymphocyte antigen 4 +49 A/G was significantly more frequent in transplanted patients infected with torque teno virus, whereas the AG genotype was more common in transplanted patients who did not have this infection. In addition, the -1661 AA and GA genotypes and -318 TC genotypes were significantly more frequent in transplanted patients infected with the virus and who had low-grade (grades I and II) acute graft-versus-host disease. Among those with grade I graft-versus-host disease, the GG genotype of the cytotoxic T-lymphocyte antigen 4+49 A/G was more frequent in transplanted patients with torque teno virus infection, whereas the AG genotype was higher in transplanted patients who did not have this infection. CONCLUSIONS: This is the first report indicating that cytotoxic T-lymphocyte antigen 4 gene polymorphism may be implicated in prevalence of torque teno virus infection after stem cell transplant. Further larger studies and evaluation of other costimulatory molecules are suggested.

Association of killer-cell immunoglobulin-like receptor genes with acute myelogenous leukaemia.

Killer-cell immunoglobulin-like receptors (KIRs) are important because of their key roles in NK cell development and function. Some KIR genes have been associated with the incidence of haematological malignancies. This study was designed to determine whether the inheritance of specific KIR genes is associated with susceptibility to acute myelogenous leukaemia (AML) in Persians living in south-western Iran. KIR genes and KIR2DS4 variants were typed by polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 patients with AML and 169 healthy controls. Our results showed 10% of patients-mostly females-were classified as M3. Flt3 mutations were detected in 26% of patients, most of whom had internal tandem duplication (ITD). The frequency of activating KIRs (aKIRs)-mainly KIR3DS1-was higher in patients, whereas inhibitory KIRs (iKIRs)-particularly KIR3DL1 and KIR2DL1-were more common among controls. The incidence of the KIR2DS4fl allele was higher among patients with non-M3 AML than controls. We also found a higher frequency of 4 or more iKIR genes in the controls and a higher frequency of 4 or more aKIR genes in the patients. Individuals with more iKIR than aKIR belonged predominantly to the control group. Individuals with the telomeric AA genotype who had inherited the KIR2DS4fl allele were more frequent in the patient group. According to our results, increased frequency of aKIRs in patients with AML may lead to the hyperactivation of NK cells against malignant cells with reduced or lack of HLA class I molecules followed by NK cell exhaustion which allow malignant cells to progress.

Aberrant peripheral blood CD4+ CD25+ FOXP3+ regulatory T cells/T helper-17 number is associated with the outcome of patients with lymphoma.

Little is known about the clinical significance of the peripheral blood CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) and T helper-17 (Th17) cells in lymphoma patients. In this study, the prognostic and clinical significance of peripheral blood Tregs and Th17 cells were evaluated in lymphoma patients during different phases. The frequency of Tregs and Th17 lymphocytes was measured by flow cytometry method in 47 classical Hodgkin's lymphoma (cHL) and 48 diffuse large B cell lymphoma (DLBCL) patients. Our results showed that the frequency of Tregs and absolute Treg count was significantly reduced in relapsed patients compared to patients at the remission phase, as well as with newly diagnosed untreated patients in both groups. Patients who reached complete remission had elevated frequency of CD4+ FOXP3+ lymphocytes, Tregs, absolute Treg count, Treg/CD4 and Treg/Th17 ratio in the cHL group and CD4+ CD25+ cells in DLBCL group. The frequency of Tregs, absolute Treg count and Treg/Th17 ratio in cHL patients and CD4+ FOXP3+ and CD4+ CD25+ cells in DLBCL patients positively associated with survival rate. Moreover, the percentage of Tregs and absolute Treg count positively correlated with white blood cell, platelet count and ESR level in cHL patients and with white blood cell count in DLBCL patients. The initial number of Tregs/Th17 cells and also the Treg/Th17 ratio was not associated with changes in disease-free survival (DFS) in both groups. Therefore, higher frequency of peripheral blood Tregs and Treg/Th17 ratio might be associated with a favorable outcome in lymphoma patients, better response to chemotherapy and lower rate of relapse.

Genetic Variation of Costimulatory Molecules, Including Cytotoxic T-Lymphocyte Antigen 4, Inducible T-Cell Costimulator, Cluster Differentiation 28, and Programmed Cell Death 1 Genes, in Iranian Patients With Leukemia.

OBJECTIVES: There are limited studies about the possible relationship between genetic variations of costimulatory genes and susceptibility to hematologic malignancies like leukemia and lymphoma. MATERIALS AND METHODS: This cross-sectional study included 59 leukemia patients. The polymorphisms of costimulatory molecules, including the CTLA-4 gene (-318 C/T, -1722 T/C, -1661 A/G, +49 A/G), PD-1 gene (1.3 A/G, 1.9 C/T), ICOS gene (1720 C/T), and CD28 gene (17 C/T), were analyzed by polymerase chain reaction-restriction fragment length polymorphism methods. RESULTS: Our results showed that the TT genotype and T allele of the CTLA-4 -318 T/C polymorphism, the AA genotype of CTLA-4 +49 A/G polymorphism, and the CT genotype of PD-1 1.9 C/T polymorphism were significantly higher in healthy controls (P < .05). However, the AG genotype of the CTLA-4 +49 A/G, the CC genotype of the PD-1 1.9 C/T, and the CT genotype of the CD28 +17C/T polymorphism were significantly increased in patients with leukemia (P < .05). When the genotype frequency of costimulatory genes was compared between different leukemia groups, we observed that the A allele of the CTLA-4 +49 A/G and the CC genotype and C allele of the CD28 +17 C/T polymorphism were significantly higher in patients with acute leukemia than in those with chronic leukemia (P < .05). Among leukemia patients, the AA genotype of CTLA-4 +49A/G polymorphism was significantly increased in patients with acute myeloid leukemia, whereas the AG genotype was more prevalent in patients with acute lymphoblastic leukemia (P < .05). CONCLUSIONS: We show for the first time that genetic variations of costimulatory molecules CTLA-4, CD28, and PD-1 may be associated with susceptibility of Iranian patients to leukemia.

Aberrant Expression of the miR-181b/miR-222 after Hematopoietic Stem Cell Transplantation in Patients with Acute Myeloid Leukemia.

Recently, dysregulated expression of various micro RNAs has been reported in hematologic malignancies, especially AML disease which affects normal hematopoiesis in these patients and thereby contribute to clinical outcome of AML patients, associated with either poor or favorable prognosis. Herein, we evaluated the expression of miR-181b and miR-222 in acute myeloid leukemia patients and correlation with response to therapy after hematopoietic stem cell transplantation. Eighty newly diagnosed AML patients and 80 healthy controls were recruited. The expression of miR-181b and miR-222 was evaluated by real-time SYBR Green PCR method. miR-181b gene expression was significantly increased (4.7 fold) whereas miR-222 was decreased (18.3 fold) in AML patients compared to controls (P = 0.03 and P < 0.001, respectively). Both miR-181b and miR-222 were not associated with response to treatment (P > 0.05). Also, miR-181b and miR-222 were not differentially expressed in AML patients with M3 compared to non-M3 FAB subtypes (P > 0.05). miR-181b and miR-222 are aberrantly expressed in AML patients and their baseline level is not associated with response to treatment.

Overexpression of indoleamine 2,3-dioxygenase correlates with regulatory T cell phenotype in acute myeloid leukemia patients with normal karyotype.

BACKGROUND: Production of immunosuppressive enzymes such as indoleamine 2,3-dioxygenase (IDO) is one of the strategies employed by hematologic malignancies, including acute myeloid leukemia (AML), to circumvent immune surveillance. Moreover, IDO has the ability to convert CD4+CD25- conventional T cells into regulatory T cells (Tregs). In this study, we evaluated the expression of IDO in cytogenetically normal acute myeloid leukemia (CN-AML) patients and its correlation with the Treg marker, FOXP3, as well as clinical and laboratory parameters. METHODS: Thirty-seven newly diagnosed CN-AML patients were enrolled in our study along with 22 healthy individuals. The expression of the IDO and FOXP3 genes was analyzed by SYBR Green real-time PCR. RESULTS: Both IDO and FOXP3 were highly upregulated in CN-AML patients compared to control groups (P=0.004 and P=0.031, respectively). A positive correlation was observed between IDO and FOXP3 expression among AML patients (r=0.512, P=0.001). Expression of IDO and FOXP3 showed no significant correlation with laboratory parameters such as white blood cell and platelet counts, hemoglobin levels, bone marrow blast percentage, gender, and FLT3 mutation status (P>0.05). CONCLUSION: Higher IDO expression in CN-AML patients may be associated with an increased Treg phenotype which may promote disease progression and lead to poor prognosis of CN-AML patients.

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics.

OBJECTIVE: In this study, we evaluated the frequency of FMS-like tyrosine kinase 3 (FLT3-ITD and FLT3-TKD) and nucleophosmin (NPM1) mutations in Iranian patients with cytogenetically normal acute myeloid leukemia (CN-AML). The clinical and laboratory characteristics were compared between wild-type and mutant cases. MATERIALS AND METHODS: Seventy newly diagnosed de novo AML patients were recruited at the time of diagnosis prior to chemotherapy; among them, 54 had CN-AML. For detecting mutations, the FLT3 and NPM1 genes were amplified by the polymerase chain reaction method, followed by direct sequencing. RESULTS: Our results showed that the frequencies of FLT3-ITD, FLT3-TKD, and NPM1 mutations in CN-AML patients were 25.9%, 5.9%, and 20.8%, respectively. The most frequent NPM1 mutation type was the type A mutation. The FLT3-ITD mutation was seen more frequently in non-M3 patients compared with M3 patients. No mutation was observed in either the FLT3-TKD or the NPM1 gene in patients in the M3 French-American-British group. There was no significant association between the presence of FLT3-ITD and NPM1 mutations in CN-AML patients (p>0.05). The frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation was higher in CN-AML patients in comparison with AML patients with cytogenetic aberrations, although the differences were not statistically significant (p>0.05). There were no significant differences in mean white blood cell and platelet counts, serum hemoglobin levels, and bone marrow blast percentages between patients with wild-type and mutant FLT3-ITD and NPM1 genes (p>0.05). No difference was observed in the frequency of FLT3-ITD or NPM1 mutation regarding age or sex (p>0.05). CONCLUSION: Given the high stability of NPM1 during the disease course, it can be used in combination with FLT3 as well as other known genetic markers to monitor patients, especially for minimal residual disease detection.

Efficacy of Deferasirox (Exjade®) in Modulation of Iron Overload in Patients with ß-Thalassemia Intermedia.

Because of insufficient erythropoiesis, peripheral hemolysis and increased gastrointestinal iron absorption, iron overload is still a matter of debate in ß-thalassemia intermedia (ß-TI) patients, which can be overcome using iron chelators. However, data on use of iron chelators in ß-TI patients is highly restricted. The aim of this study was to evaluate the efficacy of oral administration of deferasirox (Exjade(®) or DFX) by assessment of serum ferritin levels in ß-TI patients. In this quasi-experimental study, 50 ß-TI patients with serum ferritin levels >1000 ng/mL were selected and received oral DFX for 12 consecutive months. Iron overload was measured by checking serum ferritin levels every 2 months and the results were compared with the baseline level. The mean serum ferritin was decreased during 1 year of chelation therapy without any toxic effect. Although the difference between baseline ferritin and ferritin levels at the end of second month was not remarkable (p = 0.88), a significant reduction in serum ferritin was observed after 4 (p = 0.01), 6 (p = 0.001), 8 (p < 0.001), 10 (p < 0.001) and 12 months (p < 0.001) of chelation therapy compared to its baseline levels. There was no correlation between baseline ferritin levels and age (p = 0.574). In addition, no statistically significant difference was observed about change in serum ferritin levels after 6 and also 12 months of therapy between patients who had undergone splenectomy and those who did not (p = 0.796 and 0.859, respectively). Iron chelation therapy with DFX is safe and effective in reducing serum ferritin levels in ß-TI patients who suffer from side effects of iron overload.

Alteration in frequency and function of CD4⁺CD25⁺FOXP3⁺ regulatory T cells in patients with immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder characterized by production of auto-antibodies against platelet antigens. It is obvious that regulatory T cells (Tregs) have a major role in controlling immune homeostasis and preventing autoimmunity.To investigate the frequency and functions of Tregs, twenty ITP patients and twenty age- and sex-matched healthy controls were recruited. The peripheral blood mononuclear cells were isolated and the proportion of Tregs was defined by flow cytometry method. The expression of immune-regulatory markers, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and glucocorticoid induced tumor necrosis factor receptor (GITR) were also assessed by quantitative Real-time PCR TaqMan method. For evaluation of Treg function, Tregs were enriched and their ability to inhibit proliferation of T cells was measured and levels of immune-regulatory cytokines IL-10 and TGF-ß were also measured.Results showed that the frequency of Tregs and the mean fluorescence intensity of FOXP3 protein significantly decreased in ITP patients compared to those in healthy controls. In addition, there was a significant reduction in relative expression of both CTLA-4 and GITR mRNA in ITP patients (P=0.02 and P=0.006, respectively). The suppressive function of Tregs also diminished in ITP patients compared to that in controls. Both IL-10 and TGF-ß cytokines were produced in lower amounts in ITP patients than controls.It could be concluded that alteration in Treg frequency and functional characteristics might be responsible for loss of self-tolerance and subsequently destructive immune responses observed in ITP patients.