C-abl and bcr are rearranged in a Ph1-negative CML patient.

Chromosomal analysis of a patient with chronic myelocytic leukemia (CML) revealed a translocation (9;12) (q34;q21) without a detectable Philadelphia chromosome (Ph1). Using molecular approaches we demonstrate (i) a rearrangement within the CML breakpoint cluster region (bcr) on chromosome 22, and (ii) a joint translocation of bcr and c-abl oncogene sequences to the derivative chromosome 12. These observations support the view that sequences residing on both chromosome 9 (c-abl) and 22 (bcr) are involved in the generation of CML and suggest that a subset of Ph1-negative patients may in fact belong to the clinical entity of Ph1-positive CML.     

Minute chromosomes in a case of chronic Ph1-positive myeloid leukemia

Minute chromosomes (mins) were found in all bone marrow cells in a 62-year-old woman with Ph1 positive chronic myeloid leukemia. The cells had a range of 39 to 50 chromosomes with a mode of 46. The examined metaphases had a variety of numerical and structural chromosome abnormalities, as well as 2 to 6 minute chromosomes. Our case supports the assumption that there may be a correlation between the presence of mins in leukemias and the resistance of the cells to chemotherapy or the rapid progression of the disease.     

Monocyte-chemoattractant-protein-1-mediated migration of human monocytes towards blasts from patients with acute myeloid leukemia

 Purpose: In the present study the possible clinical relevance of monocyte chemoattractant protein (MCP)-1 in patients with acute myeloid leukemia (AML) was established. Methods: The pattern of migration of human monocytes towards the supernatants of blasts from 15 patients with AML was studied and the role of MCP-1, produced by these blasts, was assessed. Results: In 4 patients (group 1) the amount of monocyte migration was low and not inhibited by the addition of anti-hMCP-1. In 11 patients, the amount of monocyte migration was high; after addition of anti-hMCP-1, monocyte migration was either completely (8 patients, group 2), or partly or not (3 patients, group 3) inhibited to the level of chemokinesis. In groups 1 and 2, there was a good correlation (r=0.67) between the concentration of MCP-1 in the supernatants and the amount of monocyte migration. In group 3, such a correlation was not evident, suggesting that another chemokine might be involved or MCP-1 function was impaired by an unknown substance. Finally, measurements of MCP-1 during culture of AML blasts showed that the time at which maximal amounts of MCP-1 are produced differs between the AML samples. Conclusions: AML blasts produce different amounts of MCP-1, which plays an important role in monocyte migration towards most AML blasts. Therefore, in the context of adoptive immunotherapy, MCP-1 might be involved in future tumor vaccination programmes using autologous MCP-1-transfected irradiated AML blasts. Received: 4 May 2000 / Accepted: 26 October 2000     

RACK1 promotes the proliferation of THP1 acute myeloid leukemia cells

The receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in the regulation of multiple signaling pathways, has been reported to contribute to the survival of leukemic progenitor cells by enhancing the activity of glycogen synthase kinase 3β (GSK3β). However, it remains unknown whether RACK1 also contributes to the oncogenic growth of acute myeloid leukemia (AML) cells. Here, we report that transient or stable silencing of endogenous RACK1 expression by RACK1 short hairpin RNAs (shRNAs) led to impaired proliferation of THP1 AML cells without inducing terminal differentiation. Further exploration revealed that RACK1 loss-of-function resulted in reduced GSK3β activity. GSK3β shRNA treatment showed similar effects to RACK1 loss-of-function. Our data collectively suggest that RACK1 contributes to THP1 cell proliferation through, at least partially, enhancing GSK3β activity. Thus, targeting RACK1 may have some important therapeutic implications in the treatment of AML.     

Dendritic cells generated from acute myeloid leukemia (AML) blasts maintain the expression of immunogenic leukemia associated antigens

Recently, the focus is on new specific immunotherapies for AML such as cellular therapies employing dendritic cells (DCs) generated from AML blasts. AML-DCs express constitutionally leukemia-associated antigens (LAAs) present in AML blasts they are generated from. Here we investigated whether the generation of AML-DCs would alter the expression level of LAAs. Moreover, we evaluated the presence of HLA and costimulatory molecules on AML blasts versus AML-DCs. Quantitative real-time polymerase chain reaction (PCR) was performed for the following LAAs: preferentially expressed antigen in melanoma (PRAME), the receptor for hyaluronic acid mediated motility (RHAMM/CD168), Wilms tumor gene 1 (WT-1) and proteinase 3. The expression of HLA-ABC, HLA-DR, CD40, CD80, CD83 and CD86 was evaluated by flow cytometry. RHAMM protein expression was evaluated by immunocytochemistry, recognition of AML-DCs by PRAME epitope-specific T cells was evaluated in a chromium-release assay. Quantitative real-time PCR for AML-DCs versus AML blasts showed an alteration in mRNA expression of LAAs. An elevated PCR signal for PRAME was detected in 7/12 AML-DC preparations. 6/12 AML-DC preparations showed a significant upregulation of the PCR signal for RHAMM. A stronger WT-1 and proteinase-3 signal was observed in PCR for only 2/12 and 1/12 AML-DCs , respectively. All preparations showed a strong expression of at least one of the LAAs examined. As demonstrated by flow cytometry, AML-DCs strongly upregulated costimulatory molecules like CD40 and CD80 in comparison with AML blasts. AML-DCs tested positive for RHAMM protein. PRAME positive AML-DCs were recognized by specific T cells. AML-DCs might constitute a powerful tool in immunotherapy for AML. Real-time PCR allows a quick and quantitative assessment of immunologically relevant LAA expression with only 10⁵ DCs and might be helpful for the decision whether the AML-DC vaccination strategy is favourable or not.     

Analyzing the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays

ABSTRACT: BACKGROUND: The Real-time PCR Array System is the ideal tool for analyzing the expression of a focused panel of genes. In this study, we will analyze the gene expression profile of pediatric acute myeloid leukemia with real-time PCR arrays. METHODS: Real-time PCR array was designed and tested firstly. Then gene expression profile of 11 pediatric AML and 10 normal controls was analyzed with real-time PCR arrays. We analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis Tool. RESULTS: We designed and tested 88 real-time PCR primer pairs for a quantitative gene expression analysis of key genes involved in pediatric AML. The gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile were imported into the Ingenuity Pathway Analysis Tool. The results revealed 12 significant networks. Of these networks, Cellular Development, Cellular Growth and Proliferation, Tumor Morphology was the highest rated network with 36 focus molecules and the significance score of 41. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to hematological disease, cell death, cell growth and hematological system development. In the top canonical pathways, p53 and Huntington's disease signaling came out to be the top two most significant pathways with a p value of 1.5E-8 and2.95E-7, respectively. CONCLUSIONS: The present study demonstrates the gene expression profile of pediatric AML is significantly different from normal control; there are 19 genes up-regulated and 25 genes down-regulated in pediatric AML. We found some genes dyes-regulated in pediatric AML for the first time as FASLG, HDAC4, HDAC7 and some HOX family genes. IPA analysis showed the top important pathways for pediatric AML are p53 and Huntington's disease signaling. This work may provide new clues of molecular mechanism in pediatric AML.     

Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia

Experiments performed in mice revealed that anthracyclines stimulate immunogenic cell death that is characterized by the pre-apoptotic exposure of calreticulin (CRT) on the surface of dying tumor cells. Here, we determined whether CRT exposure at the cell surface (ecto-CRT) occurs in human cancer in response to anthracyclines in vivo, focusing on acute myeloid leukemia (AML), which is currently treated with a combination of aracytine and anthracyclines. Most of the patients benefit from the induction chemotherapy but relapse within 1–12 months. In this study, we investigated ecto-CRT expression on malignant blasts before and after induction chemotherapy. We observed that leukemic cells from some patients exhibited ecto-CRT regardless of chemotherapy and that this parameter was not modulated by in vivo chemotherapy. Ecto-CRT correlated with the presence of phosphorylated eIF2α within the blasts, in line with the possibility that CRT exposure results from an endoplasmic reticulum stress response. Importantly, high levels of ecto-CRT on malignant myeloblasts positively correlated with the ability of autologous T cells to secrete interferon-γ on stimulation with blast-derived dendritic cell. We conclude that the presence of ecto-CRT on leukemia cells facilitates cellular anticancer immune responses in AML patients.     

Effective and specific control of aml1/eto gene expression in acute myeloid leukemia cells by lentivecior-based RNA-interference

In presented work, new approach for the control of aml1/eto gene expression in t(8;21)(q22;q22)-positive acute myeloid leukemia cells has been developed. The technique is based on using the RNA-interference and lentiviral transduction methodology. Two new lentiviral vector sets for induction of constitutive anti-aml1/eto RNA-interference in acute myeloid leukemia cells have been developed and tested. The first set was based on use of artificial microRNAs (miRNAs) and second one was intended for production of short hairpin RNAs (shRNAs). It was shown that Kasumi-1 and SKNO-1 leukemia cells can be efficiency transduced by each new lentiviral vector. Moreover, the percent of modified leukemia cells that may be easily evaluated in multiplicity of infection (MOI) test achieved more than 90% for Kasumi-1 and SKNO-1 cells at MOI 40 and 20, respectively. Comparative study elucidated that the anti-aml1/eto shRNA-based approach induced a stronger knock-down of aml1/eto gene in Kasumi-1 and SKNO-1 cells than the miRNA-based method did. We hope that the proposed approach may become useful instrument for controlling the aml1/eto gene expression in vitro as well as in vivo investigations of function and biological role of the gene.     

Geographic heterogeneity of the AML1-ETO fusion gene in Iranian patients with acute myeloid leukemia

Background:

The human AML1 gene, located on chromosome 21, can be fused to the AML1- eight-twenty-one (ETO) oncoprotein on chromosome eight, resulting in a t(8;21)(q22;q22) translocation. Acute myeloid leukemia (AML) associated with this translocation is considered a distinct AML with a favorable prognosis. Due to the various incidences of the translocation, which is associated with geographic diversities, investigation of molecular epidemiology is important to increase the awareness of physicians and hematologists regarding the frequency this chromosomal aberration.

Methods:

The patients were classified according to the French–American–British classification into eight groups: M0–M7. Determination of the prevalence of the AML1-ETO fusion gene was accomplished by TaqMan real-time PCR. Bone marrow samples from 113 patients with newly-diagnosed, untreated AML -M1, -M2, and -M4, and 20 healthy controls admitted to the Ghaem Hospital in Mashhad, Iran were studied.

Results:

The AML1-ETO fusion gene was detected up 50% of the M2 subgroup and absent in the M1 and M4 subtypes and healthy controls. Comparison of the prevalence of the t(8;21) translocation with results of previous studies showed that it varies between countries. This result may be due to geographic or ethnic differences, or both.

Conclusions:

The relatively high prevalence of the t(8;21) translocation in Iran was similar to that found in other Asian countries. It was closely associated with female gender, relatively young age, and FAB-M2 subtype. Its distribution varied considerably with geographic area. Therefore, further studies are needed to provide epidemiological data important for the establishment of optimal therapeutic strategies applicable to patients of each region. Key Words: Acute myeloid leukemia, AML1-ETO, M2, Prevalence, t(8;21)     

Males with chronic myeloid leukemia and the 45, XO,Ph1 chromosome pattern

Two cases of chronic myeloid leukemia in which the cytogenetic clone was 45, XO, Ph¹ are described and compared with 20 cases recorded in the literature. The 45, XO line is peculiar to the leukemic cells and is not a manifestation of constitutional mosaicism. It probably arises from a 46, XY, Ph¹ line by loss of the Y chromosome. Because of the few cases reported, one cannot be certain that these men have a better prognosis, although the median survival time suggests that this is so. Infertility is not part of this disorder.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

Decreased expression of DNAM-1 on NK cells from acute myeloid leukemia patients

This study tested the hypothesis that the expression of CD112 and CD155 (DNAM-1 ligands) on leukemic blasts induces a decreased expression of the activating receptor DNAM-1 on natural killer (NK) cells from acute myeloid leukemia (AML) patients. DNAM-1 is a co-receptor involved in the activation of NK cell cytotoxicity after its interaction with its ligands CD112 and CD155 on target cells. Here we study the expression of DNAM-1 on NK cells and DNAM-1 ligands on blasts from AML patients stratified by age. The results demonstrate that NK cells from AML patients younger than 65 years have a reduced expression of DNAM-1 compared with age-matched controls. The analysis of DNAM-1 ligands showed a high expression of CD112 and CD155 on leukemic blasts. An inverse correlation between CD112 expression on leukemic blasts and DNAM-1 expression on NK cells was found. Furthermore, downregulation of DNAM-1 was induced on healthy donors' NK cells after in vitro culture with leukemic blasts expressing DNAM-1 ligands. In conclusion, these results support the hypothesis that receptor-ligand crosslinking downregulates DNAM-1 expression on NK cells from patients <65 years of age. Considering the relevance of DNAM-1 in NK recognition and killing of leukemic cells, the reduced expression of this receptor on NK cells from AML patients can represent an additional mechanism of tumor escape.     

Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARalpha and PLZF-RARalpha fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARalpha from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells.