Ultrasensitive quantitation of FLT3-ITD mutation in patients with acute myeloid leukemia using ddPCR.

BACKGROUND: FLT3-ITD mutations occur in 45-50% of cytogenetically normal AML patients. Conventional fragment analysis using capillary electrophoresis is routinely used to quantitate FLT3-ITD mutations. Fragment analysis however has limited sensitivity. METHODS AND RESULTS: Here, FLT3-ITD was quantified in AML patients using an in-house developed ultra-sensitive droplet digital polymerase chain reaction assay (ddPCR). The allelic ratio of FLT3-ITD was also absolutely measured by both Fragment analysis and ddPCR. The sensitivity of ddPCR in quantitation of FLT3-ITD mutation was superior to Fragment analysis. CONCLUSION: This study demonstrates the feasibility of using the described in-house ddPCR method to quantify the FLT3-ITD mutation and measure FLT3-ITD AR in AML patients.

Inhibition of Skp2 enhances doxorubicin-induced cell death in B cell precursor acute lymphoblastic leukemia.

S-phase kinase-associated protein 2 (Skp2) is a well-defined component of the Skp2-Culin1-F-box (SCF) E3 ubiquitin ligase complex, which is involved in cell cycle progression and considered a prognostic marker in cancers. Overexpression of Skp2 is frequently observed in patients with acute lymphoblastic leukemia (ALL). Inhibition of this protein may be a valuable strategy to induce apoptosis in malignant cells. Less well known is the effect of Skp2 inhibition on the potentiation of the chemotherapeutic-induced cell death in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Our results demonstrated that inhibition of the Skp2 using SZL P1-41, not only resulted in caspase-mediated apoptosis but also potentiated doxorubicin-induced apoptosis in BCP-ALL cell lines (NALM-6 and SUP-B15). SZL P1-41 in combination with doxorubicin altered cell cycle distribution and the level of cyclins and cyclin-dependent kinases in BCP-ALL cells. DNA damage response genes were also upregulated in presence of the doxorubicin and SZL P1-41 in both cell lines. In conclusion, our results indicated that inhibition of Skp2 either alone or in a combination with doxorubicin may hold promise in the future treatment of BCP-ALL.

Involvement of classic and alternative non-homologous end joining pathways in hematologic malignancies: targeting strategies for treatment.

Chromosomal translocations are the main etiological factor of hematologic malignancies. These translocations are generally the consequence of aberrant DNA double-strand break (DSB) repair. DSBs arise either exogenously or endogenously in cells and are repaired by major pathways, including non-homologous end-joining (NHEJ), homologous recombination (HR), and other minor pathways such as alternative end-joining (A-EJ). Therefore, defective NHEJ, HR, or A-EJ pathways force hematopoietic cells toward tumorigenesis. As some components of these repair pathways are overactivated in various tumor entities, targeting these pathways in cancer cells can sensitize them, especially resistant clones, to radiation or chemotherapy agents. However, targeted therapy-based studies are currently underway in this area, and furtherly there are some biological pitfalls, clinical issues, and limitations related to these targeted therapies, which need to be considered. This review aimed to investigate the alteration of DNA repair elements of C-NHEJ and A-EJ in hematologic malignancies and evaluate the potential targeted therapies against these pathways.

MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies.

MYC oncogene is a transcription factor with a wide array of functions affecting cellular activities such as cell cycle, apoptosis, DNA damage response, and hematopoiesis. Due to the multi-functionality of MYC, its expression is regulated at multiple levels. Deregulation of this oncogene can give rise to a variety of cancers. In this review, MYC regulation and the mechanisms by which MYC adjusts cellular functions and its implication in hematologic malignancies are summarized. Further, we also discuss potential inhibitors of MYC that could be beneficial for treating hematologic malignancies.

Utilization of CRISPR/Cas9 gene editing in cellular therapies for lymphoid malignancies.

The ability to change the genetic information of immune cells is a powerful tool for basic and clinical settings. CRISPR/Cas9 gene editing technology by providing an efficient approach has accelerated immune cell therapy of cancers. Lymphoid cancers comprise a wide array of disease including lymphoma, multiple myeloma and lymphocytic leukemia. Here, we review therapeutic applications of the CRISPR/Cas9 technology for immune cell therapy of common lymphoid malignancies. We describe current and future therapeutic application of CRISPR/Cas9 technology with the focus on the production and applications of engineered hematopoietic and immune cells against lymphoid malignancies. Furthermore, we provide an overview of the possible challenges and optimization of CRISPR/Cas9 system for ex- and in vivo applications within recent years.

Immune checkpoints in hematologic malignancies: What made the immune cells and clinicians exhausted!

Hematologic malignancies comprise a considerable part of cancers with high mortality at any age. Since the introduction of hematopoietic stem cell transplantation (HSCT), the overall survival of patients dramatically increased. The main goal of HSCT is the induction of a graft-versus-leukemia effect to eradicate the residual cancer cells and also reconstitute a healthy immune system for patients. However, relapse is a nettlesome challenge of HSCT. Like many other tumors, hematologic cancer cells induce immune exhaustion leading to immune escape and relapses after HSCT. Besides malignant cells, inhibitory cells such as tumor-associated macrophages and myeloid-derived suppressor cells express various inhibitory receptors capable of inducing exhaustion in immune cells, especially T and natural killer cells. The significance of immune checkpoint blocking in tumor regression in clinical trials led to the 2018 Nobel Prize in Physiology/Medicine. Here, we reviewed the clinical roles of immune checkpoints in hematologic malignancies and post-HSCT relapses.

Assessment of a poly-epitope candidate vaccine against Hepatitis B, C, and poliovirus in interaction with monocyte-derived dendritic cells: An ex-vivo study.

Design and application of epitope-based polyvalent vaccines have recently garnered attention as an efficient alternative for conventional vaccines. We previously have reported the in silico design of HHP antigen which encompasses the immune-dominant epitopes of Hepatitis B surface antigen (HBsAg), Hepatitis C core protein (HCVcp) and Poliovirus viral proteins (VPs). It has been shown that the HHP has desirable conformation to expose the epitopes, high antigenicity and other desired physicochemical and immunological properties. To confirm the accuracy of these predictions, the ex-vivo immunogenicity of the HHP was assessed. The HHP gene was chemically synthesized in pET28a and expressed in E. coli (BL21). The expressed protein was purified and its immunological potency was evaluated on dendritic cells (DCs) as antigen presenting cells (APCs). Functional analysis was assessed in co-cultivation of autologous T-cells with matured DCs (mDCs). T-cell activation, proliferation and cytokines secretion were evaluated using flowcytometry and ELISA methods. Our results indicated that the HHP could induce the DC maturation. The mDCs were able to trigger T-cell activation and proliferation. In silico design and ex-vivo confirmation of immunological potential could pave the way to introduce efficient immunogens for further analysis. The ability of HHP in DC maturation and T-cell activation makes it an amenable vaccine candidate for further in-vivo studies.

Altering chromatin methylation patterns and the transcriptional network involved in regulation of hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) are quiescent cells with self-renewal capacity and potential multilineage development. Various molecular regulatory mechanisms such as epigenetic modifications and transcription factor (TF) networks play crucial roles in establishing a balance between self-renewal and differentiation of HSCs. Histone/DNA methylations are important epigenetic modifications involved in transcriptional regulation of specific lineage HSCs via controlling chromatin structure and accessibility of DNA. Also, TFs contribute to either facilitation or inhibition of gene expression through binding to enhancer or promoter regions of DNA. As a result, epigenetic factors and TFs regulate the activation or repression of HSCs genes, playing a central role in normal hematopoiesis. Given the importance of histone/DNA methylation and TFs in gene expression regulation, their aberrations, including changes in HSCs-related methylation of histone/DNA and TFs (e.g., CCAAT-enhancer-binding protein α, phosphatase and tensin homolog deleted on the chromosome 10, Runt-related transcription factor 1, signal transducers and activators of transcription, and RAS family proteins) could disrupt HSCs fate. Herewith, we summarize how dysregulations in the expression of genes related to self-renewal, proliferation, and differentiation of HSCs caused by changes in epigenetic modifications and transcriptional networks lead to clonal expansion and leukemic transformation.

Plasma levels of norepinephrine and expression levels of ß2-adrenergic receptor gene correlate with the incidence of acute graft-versus-host disease.

Background: Acute graft-versus-host disease is a major complication in allogeneic hematopoietic stem-cell transplantation. Epinephrine and norepinephrine are stress hormones which affect many cells, including immune cells through interaction with adrenergic receptors, mainly ß2-adrenergic receptor. The immunomodulatory effects of epinephrine, norepinephrine, and signaling of the adrenergic receptor have been shown to decrease the probability of the acute graft-versus-host disease in animal models. The aim of our study was to investigate the possible correlations between the serum levels of epinephrine and norepinephrine and also leukocytic expression levels of ß2-adrenergic receptor with the incidence of acute graft-versus-host disease in patients undergoing allogeneic hematopoietic stem-cell transplantation. Methods: In this study, the plasma levels of epinephrine and norepinephrine and the leukocytic expression of ß2-adrenergic receptor gene were measured and compared in allogeneic hematopoietic stem-cell transplantation patients with and without acute graft-versus-host disease. Data were analyzed and illustrated using SPSS 19 and GraphPad Prism 6. The student T-test, Pearson, and Spearman's tests were performed and p<0.05 was considered as significant. Results: We showed that the plasma levels of norepinephrine and the relative amount of the mRNA of ß2-adrenergic receptor at 7 and 21 days after allogeneic hematopoietic stem-cell transplantation were significantly lower in patients with acute graft-versus-host disease than recipients without acute graft-versus-host disease. There were also negative correlations between the plasma levels of norepinephrine, leukocytic levels of the mRNA of ß2-adrenergic receptor, and the incidence of acute graft-versus-host disease. Conclusion: Our results suggest that stress hormones and their receptor might have a role in preventing acute graft-versus-host disease and could be promising factors in controlling the outcome of allogeneic hematopoietic stem-cell transplantation.

Effects of ABO incompatibility on the outcome of allogeneic hematopoietic stem cell transplantation.

BACKGROUND: ABO compatibility between donor and recipient is no necessary in allogeneic hematopoietic stem cell transplantation (AHSCT). Incompatible transplantations can be divided into three groups based on the donor and recipient blood groups. The influence of each kind of incompatibilities on the outcome of patients does not seem to be consistent. This study aimed to investigate the outcome of AHSCT patients focusing on compatibility statues. METHOD: This retrospective study was conducted on 186 patients who underwent first AHSCT, includes 108 identical, 38 minor, 32 major and eight bidirectionalABO incompatible recipients. Comparative analysis was performed for common clinical transplantation outcomes. RESULTS: There was no statistically significant association betweenABO incompatibility and graft-versus-host disease, WBC or platelet engraftment, and transfusion requirement. WBC engraftment rate was significantly lower in minor-incompatible patients. Furthermore, total and direct bilirubin which (the hemolysis biomarkers) were considerably higher in the bidirectional incompatible group, compared to the other patients. CONCLUSION: Our results indicate that theABO incompatibility might be an effective factor in engraftment time and laboratory hemolysis. Elucidating the impact of ABO incompatibility on the clinical outcome of patients warrants an extended and deep investigation in a large-scale study with comprehensive variables such as survival, relapse, and other complication of transplantation.

Evaluation of H3 histone methylation and colony formation in erythroid progenitors treated with thalidomide and sodium butyrate.

OBJECTIVES: ß-thalassemia and sickle cell disease are hemoglobinopathies with reduced/absent ß chains in the former and dysfunctional ß chains in the latter. In both conditions, up-regulation of hemoglobin F through demethylation can alleviate the symptoms. This can be attained with drugs such as thalidomide and sodium butyrate. MATERIALS AND METHODS: This study was performed on erythroid progenitors derived from CD133+ cord blood stem cells. Erythroid progenitors were treated with thalidomide and sodium butyrate in single and combined groups. Colony-formation potential in each group was evaluated by the colony assay. Real-time polymerase chain reaction (RT-PCR) was used to evaluate the effect of these drugs on histone H3 lysine 27 (H3K27) methylation patterns. FINDINGS: Compared to other treatment groups, CD133+ cells treated with thalidomide alone produced more hematopoietic colonies. Thalidomide alone was also more effective in decreasing H3K27 methylation. CONCLUSIONS: Thalidomide shows superiority to sodium butyrate as a hypomethylating agent in this cell culture study, and it has the potential to become conventional treatment for sickle cell disease and ß-thalassemia.

Functional analysis of three recombinant A1-VWF domain mutants in comparison to wild type and plasma-derived VWF facilitates subtyping in type 2 von Willebrand disease.

Phenotypic diagnosis of VWD, in particular type 2, is challenging. Molecular diagnosis may fail to provide clarity since mutations within a short stretch of the same domain may cause various phenotypes, and since even experts will ascribe different subtypes to similar mutations. We assessed diagnostic difficulty in VWD by investigating five cases where phenotypic data was unclear. We identified 3 novel mutations within the A1 domain of the VWF gene: L1460F (2 related patients), Y1363C (1 patient), E1389K (2 related patients). These were not found in 100 normal individuals or documented in the VWF mutation database. Detailed functional analysis of recombinant mutants included VWF multimers, VWF:Ag, VWF:RCo, VWF:CB, and Platelet-VWF binding studies, and results assessed against recombinant WT and plasma derived (pd) VWF. Multimer analysis showed clear loss of HMW VWF with E1389K only, consistent with coincident low relative CB/Ag ratio. VWF-platelet binding studies using two independent approaches showed enhanced activity for L1460F, but reduced activity for E1389K and Y1363C. A novel finding was that WT rVWF showed enhanced platelet binding in RIPA analysis compared to pdVWF with this being dependent on the dilution material used. Through these extensive studies, we assigned L1460F to type 2B, E1389K to 2A, and Y1363C to 2M VWD. Thus, although molecular analysis is not required to classify VWD patient subtypes, a thorough and combined phenotypic, genotypic and functional analysis will assist assignment of the VWD subtype.

Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice.

The use of plasma-derived factor VIII (pdFVIII) concentrates in hemophilia A has been reported to result in reduced anti-FVIII antibody formation. In this study, we have investigated whether the cytokine microenvironment induced by pdFVIII has an influence on reducing anti-FVIII antibody titers in hemophilic mice. Microarray and confirmatory quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments show that pdFVIII infusion causes a different transcriptional profile in dendritic cells than recombinant FVIII (rFVIII). Both treatments caused up-regulation of proinflammatory gene expression, but rFVIII and pdFVIII treatments promote expression of genes that induce Th1 and Th2 responses, respectively. Moreover, administration of rFVIII or pdFVIII concentrates resulted in distinct T-cell splenic cytokine microenvironments. rFVIII induced the release of Th1 cytokines and IL-10, whereas pdFVIII induced the release of Th2 cytokines and transforming growth factor-beta. We have also observed high titers of anti-human von Willebrand factor (VWF) antibodies in the pdFVIII-treated mice and propose that this results from antigenic competition. We further investigated the role of this phenomenon using infusions of FVIII and increasing concentrations of recombinant human factor IX (FIX). These studies show an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies report new mechanisms that contribute to reduced anti-FVIII antibody development in hemophilia A after pdFVIII infusions.

Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response.

Non-Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4+CD25+ regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4+CD25+ cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4+CD25+ CTLA-4+ and CD4+CD25+mTGF-beta1+ cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile.

Molecular genetic testing of hemostasis and thrombosis in developing countries: achievements, hopes, and challenges.

In today's developing world, how do we define a "developing country"? What level of effort and resources is invested in diagnosis, patient care, and research in those countries that we define to be developing? In particular, what is the situation with respect to molecular genetic testing in these countries? How much has been achieved to date, and what are the challenges to further achievements? This article describes the current status, challenges, and future hopes with respect to molecular genetic testing in hemostasis and thrombosis from the perspective of experts from three countries: Brazil, Colombia, and Iran. These individuals have lived and practiced genetic testing in their countries and have also had the experience to work and/or interact with the developed world to enable an appreciation of the difference.