Potential Effects of the FLT3-ITD Mutation on Chemotherapy Response and Prognosis of Acute Promyelocytic Leukemia.

PURPOSE: To evaluate the influence of FLT3-ITD mutations on the treatment response and long-term survival of newly-diagnosed patients with acute promyelocytic leukemia (APL) treated with all-trans retinoic acid and arsenic trioxide. METHODS: The long-term survival of 90 newly-diagnosed APL patients (age range 12-75 years) was retrospectively analyzed.The FLT3-ITD mutation rate was assayed by polymerase chain reaction (PCR) amplification and sequencing analysis. Its impact on the treatment response, event-free survival(EFS), or overall survival(OS) was investigated in patients with and without the mutations. RESULTS: The FLT3-ITD mutation rate in newly-diagnosed APL patients was 20% (18/90). The white blood cell (WBC) count at diagnosis in patients with mutations was significantly higher than that in patients without mutations while the FLT3-ITD mutation rate was higher in the high-risk group than in the low/intermediate-risk group. Patients with mutations had a significantly higher early death (ED) rate (16.67% vs 1.39%) for those lacking the mutation (P =0.024). However, the complete remission (CR) and differentiation syndrome (DS) rates in the two groups were similar. Kaplan Meier analysis for EFS and OS at five years showed a significant difference between the patients stratified by FLT3-ITD mutation status (log-rank P =0.010 and P =0.009, respectively). CONCLUSION: FLT3-ITD mutations can be related to high peripheral WBC counts in APL patients. APL patients with mutations displayed a higher ED rate compared to those without mutations. Patients carrying mutations had reduced five-year EFS and OS rates. Thus, reducing the overall death rate during induction treatment might be an effective way to improve the prognosis of patients with FLT3-ITD mutations.

Hyperfibrinolysis Is an Important Cause of Early Hemorrhage in Patients with Acute Promyelocytic Leukemia.

BACKGROUND The objective of the current study was to guide the early clinical treatment strategies by assessing the recovery of abnormal coagulation in acute promyelocytic leukemia (APL) patients during induction therapy. MATERIAL AND METHODS Retrospective analysis was performed in 112 newly-diagnosed patients with APL during induction treatment. RESULTS The early death (ED) rate in our study was 5.36% and the main cause was fetal hemorrhage. The presence of bleeding symptoms was significantly correlated with low platelet and fibrinogen levels. The values of white blood cell (WBC), lactate dehydrogenase (LDH), prothrombin time (PT), fibrinogen, and bone marrow leukemic promyelocyte in the high-risk group were significantly different from those in the low/intermediate-risk groups. Coagulation variables significantly improved after dual induction therapy. No significant difference was found in changes of platelet (PLT), prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimers, and fibrinogen among different risk groups after induction therapy. D-dimer levels were initially high and remained well above normal after 4 weeks of induction therapy. CONCLUSIONS Aggressive prophylactic transfusion to maintain high platelet and fibrinogen transfusion thresholds could reduce hemorrhage in APL patients. Immediately starting induction therapy effectively alleviated coagulopathy in APL patients. Hyperfibrinolysis was a more important event in the APL hemorrhagic diathesis.

Low platelet count is potentially the most important contributor to severe bleeding in patients newly diagnosed with acute promyelocytic leukemia.

The objective of the current study was to provide more appropriate therapeutic strategies for reducing severe hemorrhaging by assessing the recovery of abnormal coagulation indexes in patients with acute promyelocytic leukemia (APL) during induction therapy. Retrospective analyses of 112 patients newly diagnosed with APL were performed during initial treatment. In our study, the early death rate was 5.36%. Hemorrhage was the leading cause of death during the induction period (4/6). The values of white blood cell count, lactate dehydrogenase, prothrombin time (PT), fibrinogen (Fbg), hemoglobin, and bone marrow leukemic promyelocytes were significantly different in the high-risk group compared to the low/intermediate-risk groups. There were significant differences in the white blood cell count, bone marrow leukemic promyelocytes, platelet (PLT) count, and the levels of lactate dehydrogenase, d-dimer, PT, and Fbg, as well as in FLT3-ITD mutations between patients with major bleeding and those with minor bleeding. Hemostatic variables significantly improved over time during induction therapy. The recovery times of the PLT, PT, and Fbg values were significantly slower in patients with major bleeding than in those with minor bleeding. Specifically, the PLT level in patients with major bleeding was not similar to that in the minor bleeding group until after 4 weeks of treatment. Hemorrhages were the most common cause of induction death in this study. High-risk patients were more prone to serious clinical bleeding symptoms. Patients with major bleeding had more rapid proliferation characteristics and an increased incidence of FLT3-ITD mutations compared to patients with minor bleeding. Hemostatic variables recovered significantly more slowly in patients with major bleeding than in those with minor bleeding. Active induction therapy and blood product infusion are effective in preventing severe bleeding. Our results suggested that low PLT count might be the leading cause of fatal bleeding in patients newly diagnosed with APL.

[Clonal evolution in leukemia].

The theory of evolution of tumor cell population has been established for nearly 40 years. It was widely accepted for research and clinical anti-tumor treatment. Recently, it was suggested that cancer stem cells are the unit of evolution. Considering recent advances on genesis of tumor and leukemia with ecological and evolutionary views, this article reviews origin and evolution of leukemia stem cells. Over the last few years, clinical and experimental data suggest there are two paths for the origin of leukemia stem cells: from a transformed hematopoietic stem cell or progenitor. The mechanisms of leukemia stem cell formation and clonal evolution were elucidated. Sub-clonal mutations and clonal architectures in leukemia were studied and a mosaic evolution pattern is described. Random evolution or non-inherited mutations of leukemia cells would accelerate the progression of malignant disease. Finally, the mosaic or network mechanism for leukemogenesis is also discussed.

[Membrane-bound cytokine and feedforward regulation].

Feedback and feedforward widely exist in life system, both of them are the basic processes of control system. While the concept of feedback has been widely used in life science, feedforward regulation was systematically studied in neurophysiology, awaiting further evidence and mechanism in molecular biology and cell biology. The authors put forward a hypothesis about the feedforward regulation of membrane bound macrophage colony stimulation factor (mM-CSF) on the basis of their previous work. This hypothesis might provide a new direction for the study on the biological effects of mM-CSF on leukemia and solid tumors, and contribute to the study on other membrane bound cytokines.

[Co-evolutionary analysis on strategy for therapy of spreading cancer].

Evolutionary medicine can give rational explanation for metabolism diseases via ecology and evolutionary theory. Recently, the view of somatic cell macroevolution was used in the study on the genesis and development of tumors, which provided new insight in the research work on tumors. In this article, the well-adopted tumor therapy strategy, "Dancing with Cancer", was analyzed preliminarily from the point of co-evolution game theory, based on the non-classical immunology theory and genome theory. The importance of increasing host fitness by changing host life-style to enhance tolerance was emphasized, which is the basis of the Dancing with Cancer strategy. On the other hand, the spreading tumor cells are not equally malignant and spreading tumors should be treated as other chronic diseases. Finally, basic and clinical research should be strengthened to improve the efficiency of the "Dancing with Cancer" strategy.

[Mechanism of leukemia relapse: novel insights on old problem].

Relapse, which puzzled several generations of hematologists, is the bottle-neck of radical treatment for leukemias. The progress of Human Microbiome Project at the beginning of 21st century suggested that human body was a super-organism constituted by the core of human cells and symbiotic microorganisms. The elucidation and characterization of endogenous retrovirus and prion protein suggested the possible effects of co-evolutional microorganisms on human health. Recently, the elucidation of the roles of tunneling nanotubes in intercellular communication and transportation suggested a novel way for cellular communication and transport of oncogenic materials. The role and significance of in vivo cell fusion have been studied in more detail. On the other hand, donor cell leukemia was reported. All of these approaches provide novel insights for studying the mechanism of leukemia relapse. Based on previous work, the authors suggest the hypothesis: there are two possible mechanisms for the relapse of leukemias: the minimal residual disease (MRD) and intercellular transportation of oncogenic materials.

[Leukocyte synapse: structure, function and significance].

Neuronal synapse is the critical structure of neuronal network. Immune system is mainly consisted of invisible network. Recently, evidence showed that leukocyte synapses between immune cells named as immunological synapses (IS), were formed under some functional conditions to form temporal local network. In fact, they are dynamic structures, which can be classified into synapse and kinase. Different leukocytes have different synapses. Inflammatory and leukemic cells showed special patterns of IS. Similar structure is also observed in some viral infected lymphocytes, which is called virological synapse (VS). This is one of the mechanisms for viral transmission, not only enhancing the transmission efficiency but also mediating the escape from antibody neutralization, leading persistent infection. Recently the flower-like poly synapses was reported by French scientists. This is a multi-tunneling nanotube flower-like structure on cell surface. We had observed this kind of structure in EB virus infected human leukemic cell line J6-2. In this paper, the structure and function of leukocyte synapses are reviewed combined with authors' own work. Their significance is discussed.

[Latent infection of human herpes virus in hematopoietic system].

Up to date, eight types of human herpes viruses have been identified, all of which are ubiquitous, and usually establish latent infection in the host after primary infection. Since most of the herpes viruses are maintained in an asymptomatic form, they are often neglected. However, under some circumstances, these herpes viruses can cause fatal or severe diseases. Furthermore, the association of herpes viruses with hematopoietic malignancies is attracting researchers' attention. With the extensive development of hematopoietic stem cell and organ transplantation, reports regarding transplantation failure and complication caused by infection of human herpes virus has been increasing. Cytokine storm was firstly suggested as the mechanism of graft-versus-host diseases. In recent years, which has also been applied in the pathogenesis research of inflammation, and is supposed to play an important role in severe virus infection. In this paper, through discussing the possible role of latent infection of human herpes virus in the failure or complication of bone marrow or hematopoietic stem cell transplantation, and in refractory leukemia, the function and significance of latent infection of human herpes virus and the cytokine storm it caused were investigated.

[Leukemia stem cells and their microenvironment--editorial].

As pioneer of tumor stem cell research, leukemia stem cell research has not only important theoretical significance, but also clinical application potential. The survival and development of stem cells are directly impacted by their microenvironment. The research on leukemia stem cells and their microenvironment are now becoming a hot topic. The author presumes that stem cells are a population with heterogenecity and hierarchy; any single cell from the population is difficult to form a clone; the interaction between the leukemia stem cell and its microenvironment can be described by the concept of leukemia stem cell niche. In this article, the leukemia cell population with heterogenecity and hierarchy as well as leukemia stem cell niche were summarized and discussed.

[Observation on therapeutic effects of combined acupuncture and medicine therapy and simple medication on renal hypertension of chronic kidney disease].

OBJECTIVE: To probe into a method for increasing clinical therapeutic effect on renal hypertension of chronic kidney disease. METHODS: One hundred and fifty-two cases were randomly divided into a combined acupuncture and medicine group and a medication group, 76 cases in each group. The combined acupuncture and medicine group were treated with acupuncture balance points "Jiangya" and "Shenbing" as main, combined with small dose of hypotensor, Irbesartan; and the medication group were treated with oral administration of Irbesartan and Fosinopril. Their clinical therapeutic effects were compared. RESULTS: After treatment of 4 weeks, 56.58% of the patients in the combined acupuncture and medicine group reached to the objective value [DBP < or = 84.96 mm Hg (1 mm Hg= 0.133 kPa)], and 53.95% of the patients in the medication group reached to the objective value, with no significant difference between the two groups (P > 0.05). After treatment of 8 weeks and 24 weeks, the blood pressure-decreasing effect in the combined acupuncture and medicine group was better than that of the medication group (P < 0.01). After treatment, protein in the urine decreased and blood creatinine reduced, the clearance rate of endogenous creatinine raised in the two groups, with very significant differences (P < 0.01). CONCLUSION: Acupuncture combined with small dose of medicine and simple medicine have a same therapeutic effect on renal hypertension of chronic kidney disease, but with prolongation of treatment time, the therapeutic effect and advantages of the combined acupuncture and medicine therapy were superior to the medication.

[Cloning and functional analysis of P2X7 receptor from J6-1 leukemia cells].

OBJECTIVE: To clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells. METHODS: The entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope. RESULTS: The entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation. CONCLUSIONS: The entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

CD 39-associated high ATPase activity contribute to the loss of P 2 X 7-mediated calcium response in LCL cells.

The P 2 X 7 nucleotide receptor is an adenosine 5'-triphosphate (ATP)-gated ion channel, which induces cation channel opening imparting significant permeability to Ca(2+), and is widely expressed in cells of hematopoietic origin. Our previous report showed that P 2 X 7-mediated calcium response was absent in three Epstein-Barr virus (EBV)-positive and P 2 X 7 positive cell lines. In this report, we detected the cell surface ATPase activity, which contributes to the hydrolysis of extracellular ATP, and the expression of CD 39, which is the main source of ATPase on hematopoietic cells, in these cell lines. Then, we tried to restore the P 2 X 7-mediated calcium response in LCL-H and J 6-1 cells by either increasing the concentration of agonist or suppressing the ATPase activity by betagammaMeATP, a synthetic poorly metabolizable ATP analogue. The results showed that LCL-H and J 6-1 cells had higher levels of ATPase activity and CD 39 expression. The treatment of 300 microM betagammaMeATP efficiently inhibited the ATPase activity on LCL-H and J 6-1 cells. Both elevation of agonist concentration (10mM ATP or 1mM BzATP) and pretreatment with 300 microM betagammaMeATP followed by stimulation with normal concentration of agonists (1mM ATP or 0.1mM BzATP) could cause P 2 X 7-mediated calcium response in LCL-H but neither in J 6-1 cells. These results suggested that multiple mechanisms contributed to the loss of the P 2 X 7-mediated calcium response. CD 39-associated high ATPase activity contributed to the loss of the P 2 X 7-mediated calcium response in LCL-H cells, while additional mechanism(s) existed in J 6-1 cells.

Effects of various inducers on the expression of P2X7 receptor in human peripheral blood mononuclear cells.

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.

LL-37 enhances adaptive antitumor immune response in a murine model when genetically fused with M-CSFR (J6-1) DNA vaccine.

DNA vaccine against M-CSFR(J6-1) (macrophage colony-stimulating factor receptor cloned from the J6-1 leukemic cell line) has shown both protective and therapeutic effects. In this study, to explore the adjuvant effects of LL-37 to M-CSFR(J6-1) DNA vaccines, we constructed genetically fused vaccines encoding M-CSFR(J6-1) and LL-37(pF). After immunizing BALB/c mice, specific humoral and cellular immune responses were detected. Compared with pR (encoding the extracellular region of M-CSFR(J6-1)), pF was more effective in inducing humoral and cytotoxic immune response, prolonging survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells, and inducing IFN-gamma and IL-4 release by splenocytes. In this study, we also constructed pLL37 (encoding the mature LL-37) and coadministrated pLL37 and pR to see whether the genetic fusion was necessary. We found that compared with pR alone, pLL37+pR could not prolong survival of mice challenged with SP2/0-CSFR(J6-1) tumor cells. Our results suggest that when genetically fused with M-CSFR(J6-1), LL-37 could enhance adaptive immune response against M-CSFR(J6-1) in a murine model challenged with tumor cells bearing M-CSFR(J6-1).

Marked reduction of LL-37/hCAP-18, an antimicrobial peptide, in patients with acute myeloid leukemia.

We detected LL-37/hCAP-18 expression in the peripheral blood smears of 50 healthy donors and 143 patients with various hematological diseases. Compared with that in the healthy donors, expression of the protein in the neutrophils was significantly lower in patients with acute myeloid leukemia (AML), especially those with infection, but no significant difference was detected in messenger RNA level. We did not detect increased LL-37/hCAP-18 protein expression in U937 cells treated with lipopolysaccharide or Staphylococcus aureus Cowan strain. Furthermore, LL-37/hCAP-18 protein production was not restored in differentiated myeloid cell lines NB4 or HL-60 induced by all-trans retinoic acid. LL-37/hCAP-18 has been shown to play a role in host defense, and its deficiency in AML may be one of the explanations for susceptibility to infection among these patients.

[Apoptosis or necrosis, should which be expected for tumor cells?].

Evidence has indicated that low doses of anti-tumor regimens can induce cell apoptosis in vitro, although different regimens induce apoptosis by different mechanism and pathway. In recent years, new tumor treatment strategy has been mainly focused on inducing tumor cell apoptosis. The present review discusses the advantages and disadvantages of inducing tumor cell apoptosis. The benefit of inducing apoptosis is not to cause inflammatory reaction, but as its disadvantage, it inhibits immune responses, and the phagocytosis of apopotic bodies may result in horizontal transfer of genes (including oncogenes and other oncogenic materials), which can be one of the causes of tumor relapse. This paper proposes that the tumor treatment strategy should be turn into promoting tumor cell necrosis and inducing anti-tumor immune responses.

Expression of LL-37/hCAP-18 gene in human leukemia cells.

LL-37/hCAP-18 is an important part of host defense. Several diseases in human are characterized by impairment in the function of LL-37/hCAP-18 peptide. We examined the expression of LL-37/hCAP-18 in a panel of hematopoietic cell lines representing multiple cell lineages. LL-37/hCAP-18 expression at mRNA level was detected and varied among six of nine cell lines. The level of Raji cells was about eight folds higher than that of Ramos cells. However, only two cell lines, J6-1 and U937, expressed protein products. We also investigated LL-37/hCAP-18 protein in nine leukemia and three idiopathic thrombocytopenic purpura (ITP) patients via immunocytochemical staining. The rate of LL-37/hCAP-18 positive cells ranged from 60 (ITP) to 0.5% (M5). These data suggested that the low translation efficiency of LL-37/hCAP-18 expresses in some leukemia cells might be one of the reasons that leukemia patients were susceptible to infection.

Co-immunization with M-CSFR and mM-CSF DNA vaccines is better than M-CSFR-mM-CSF fusion DNA vaccine.

BACKGROUND AND OBJECTIVES: DNA vaccine against macrophage colony-stimulating factor receptor (M-CSFR) has shown both protective and therapeutic effects. In this study, we explore the possibility of using DNA vaccines against both M-CSFR and membrane-bound macrophage colony-stimulating factor (mM-CSF) to achieve better effects. DESIGN AND METHODS: Three plasmids were constructed by inserting either extracellular and transmembrane region of mM-CSF (pM), or extracellular region of M-CSFR (pR), or extracellular region of M-CSFR linked with extracellular and transmembrane regions of mM-CSF by a (Gly Gly Ser)2 flexible linker (pF), into pcDNA3.1. A SP2/0 cell line stably expressing pF (SP2/0-F) was established to evaluate humoral and cytotoxic immune responses as well as therapeutic and preventive effects induced by pM, pR, pF or pM+pR vaccination in BALB/c mice. The mechanisms of these vaccinations were also studied by monitoring the release of interleukin (IL)-4 and interferon (IFN)-g by splenocytes upon activation. RESULTS: Vaccination against two epitopes had better effects than against a single epitope while vaccination by pM+pR had the greatest effects on inducing humoral and cytotoxic immune responses, prolonging survival of mice challenged with SP2/0-F, and inducing IL-4 and IFN-g release by splenocytes. INTERPRETATION AND CONCLUSIONS: Our results suggest that co-immunization of M-CSFR and mM-CSF DNA vaccines is better than M-CSFR-mM-CSF fusion DNA vaccine.

Clone and expression of mutant M-CSF and its receptor from human leukemic cell line J6-1.

Macrophage colony-stimulating factor (M-CSF) plays important roles in hematopoietic and immunologic systems. Some isoforms or mutations have been demonstrated including membrane-bound and cellular M-CSF, which associated with some leukemia, lymphoma and other solid tumors. We previously reported that the M-CSF-like membrane-associated factor (MAF-J6-1) and its receptor was found from human leukemic cell line J6-1. In this report, the cDNA of MAF-J6-1 and its receptor were cloned. The cDNA sequence of MAF-J6-1 shows a 768bp open reading frame (ORF) with 99.2% homology to m-M-CSF, but six site mutations, including two synonymous mutations and four missense mutations. The cDNA of MAF-J6-1-R has a 2916bp ORF shared 99.6% homology with M-CSF-R, but 13 site mutations, including six synonymous mutations and seven missense mutations. At the same time, a 1662bp mutant s-M-CSF cDNA, which has 10 site mutations including three synonymous mutations and seven missense mutations, was cloned from J6-1 cells. The cDNAs of MAF-J6-1 and MAF-J6-1-R were inserted into a mammalian expression plasmid pTARGET and were expressed in COS-7 cells that demonstrated by their specific MAb. COS-7 cells transfected with MAF-J6-1-R show obvious protein tyrosine kinase (PTK) activity. Our present work shows that MAF-J6-1 and its receptor are mutations of M-CSF and its receptor.