Definition of miRNAs expression profile in glioblastoma samples: the relevance of non-neoplastic brain reference.

Glioblastoma is the most aggressive brain tumor that may occur in adults. Regardless of the huge improvements in surgery and molecular therapy, the outcome of neoplasia remains poor. MicroRNAs are small molecules involved in several cellular processes, and their expression is altered in the vast majority of tumors. Several studies reported the expression of different miRNAs in glioblastoma, but one of the most critical point in understanding glioblastoma miRNAs profile is the comparison of these studies. In this paper, we focused our attention on the non-neoplastic references used for determining miRNAs expression. The aim of this study was to investigate if using three different non-neoplastic brain references (normal adjacent the tumor, commercial total RNA, and epileptic specimens) could provide discrepant results. The analysis of 19 miRNAs was performed using Real-Time PCR, starting from the set of samples described above and the expression values compared. Moreover, the three different normal RNAs were used to determine the miRNAs profile in 30 glioblastomas. The data showed that different non-neoplastic controls could lead to different results and emphasize the importance of comparing miRNAs profiles obtained using the same experimental condition

Myelodysplastic syndromes: the pediatric point of view.

Myelodysplastic syndromes (MDS) are clonal disorders of the multipotent hematopoietic stem cell characterized by ineffective hematopoiesis and associated with marrow hypercellularity, increased intramedullary cell death and peripheral cytopenias of varying severity. Patients with myelodysplasia have a propensity (20% to 30% of cases) to undergo transformation into acute myeloid leakemia (AML), and a large body of evidence indicates that MDS represent steps in the multiphasic evolution of AML. Progression of the disease is characterized by expansion of the abnormal clone and inhibition of normal hematopoiesis leading to deterioration of the blood cell count and/or development of AML. MDS are relatively unusual in childhood, representing only 3% of pediatric hematological malignancies, although it has been reported that up to 17% of pediatric AML cases may have a previous myelodysplastic phase. The first systematic attempt at morphological classification of MDS was provided by the French-American-British (FAB) group. However, the FAB classification of MDS is only partially applicable in children. Some variants are extremely rare or absent (refractory anemia with ring sideroblasts and chronic myelomonocytic leukemia), and other peculiar pediatric disorders, represented by juvenile chronic myelogenous leukemia (JCML) and the monosomy 7 syndrome, are not included. Moreover, since there is a partial overlap between pediatric MDS and myeloproliferative disorders and the variants occurring in young children have rather specific features, some confusion still surrounds the nosographical definition of childhood MDS, so that none of the proposed classifications are widely accepted and used. Characteristically, some genetic conditions such as Fanconi's anemia, Shwachman's and Down's syndromes predispose to the development of MDS in childhood. The most common variants of childhood MDS are represented by JCML and the monosomy 7 syndrome, both disorders typically occurring in young children. JCML is characterized by a spontaneous growth of granulocyte-macrophage progenitors that show a striking hypersensitivity to granulocyte-macrophage colony-stimulating factor. Clinical presentation resembles that of some myeloproliferative disorders, with massive organomegaly usually not observed in the classically reported variants of MDS. Clinical features of the monosomy 7 syndrome resemble those observed in JCML and a differential diagnosis between these two entities relies upon the higher percentage of fetal hemoglobin, the more pronounced decrease in platelet count and, in some cases, the lack of the peculiar cytogenetic abnormality in the latter. With the number of children being cured of cancer constantly rising, a significant increase in secondary or chemotherapy-related myelodysplasia is being observed, and these disorders represent a formidable challenge for pediatric hematologists due to their poor response to chemotherapy. As a matter of fact, owing to their biological heterogeneity and aggressive clinical course in childhood, all MDS variants pose serious difficulties for successful management. If a compatible donor is available, allogeneic bone marrow transplantation (BMT) becomes the treatment of choice and should be performed during the early stages of the disease. Supportive therapy, differentiative treatments and low-dose chemotherapy, while valuable alternative therapeutic options in adults, have limited application in pediatric patients. The role of intensive chemotherapy and autologous BMT has not yet been clearly defined, and the use of hematopoietic growth factors does not seem to have a significant influence on the natural history of the disease. In the future, new insights into the events leading to progressive genetic changes in the clonal population and into the molecular basis of these genetic lesions could result in interesting new therapeutic approaches directed either at the oncogenes involved in the pathogenesis of the disease, or at the cytokines and/or their receptors causing the abnormal differentiation and proliferation of the myelodysplastic clone

NUP98-fusion transcripts characterize different biological entities within acute myeloid leukemia: A report from the AIEOP-AML group.

In the last years, collaborative studies have joined to link the degree of genetic heterogeneity of acute myeloid leukemia (AML) to clinical outcome,1, 2 allowing risk stratification before therapy and guiding post-induction treatment of children with AML. So far, still half of these patients, whose disease is usually characterized by a grim prognosis, lack a known biomarker offering opportunities of targeted treatment

Impact of N-myc amplification on median survival in children with neuroblastoma

Background: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years, and it is characterized by wide clinical and biological heterogeneity. N-myc oncogene amplification is considered to be one of the most important prognostic factors used to evaluate survival in these patients. Objectives: The aim of our study was to determine amplification of the N-myc oncogene using real-time quantitative polymerase chain reaction (PCR) and to show the influence of N-myc amplified tumors on the overall survival rate. Patients and Methods: This study is an analytical historical cohort study of forty children with neuroblastoma admitted to the Shafa Hospital, Iran from 1999 to 2010. Paraffined blocks of tumoral tissue were analyzed for N-myc amplification by a PCR. The degree of N-myc amplification was derived from the ratio of the N-myc oncogene and the single copy reference gene, NAGK. In the statistical analysis, a Kaplan-Meier survival analysis was used. Results: We found a variable degree of N-myc amplification, from 3 to 2 200, in 32 of the 40 neuroblastomas (80%). NMYC amplification was seen more frequently in patients older than 2.5 years (71.9%), stage 4 (65.6%) and female (53.1%). Median survival time in the males was significantly longer than in the females (P = 0.03). The overall median survival for N-myc amplified tumor patients was 20 months, and 30 months for the non amplified tumors. Conclusions: The N-myc amplified tumors may increase the probability of more aggressive behavior and rapid tumor progression, especially in advanced stages of neuroblastoma. This study confirmed the importance of obtaining correct measurements of oncogene amplification in the early evaluation of neuroblastomas in order to target more aggressive therapies in patients with a higher risk of cancer progression

Identification of the B-cell tumor-specific molecular fingerprint using non-radiolabelled PCR consensus primers

Abstract BACKGROUND: The complementarity determining region 3 (CDR3) of the immunoglobulin (Ig) heavy chain variable region (VH) is the most reliable molecular fingerprint for most if not all human B cells. The nucleotide sequence encoding for any B-cell tumor-specific VH CDR3 is currently identified by PCR sequencing based on procedures involving the usage of either radioactive materials, patient/family-specific primers, or bacterial cloning. PATIENTS AND METHODS: In six consecutive patients with follicular lymphoma we assessed the feasibility of a method that allows for identification of the tumor-specific VH CDR3 using consensus primers while avoiding both radioactive materials and bacterial cloning procedures. RESULTS: The tumor-specific VH CDR3 was successfully identified in all six patients in nearly half the time typically required by any other method currently utilized. The feasibility of the proposed method was not significantly affected either by the tumor-specific Ig isotype, or by the tumor infiltration in the original biopsy specimen. In the three patients for whom tumor specimen-derived hybridomas were available, the tumor-specific VH CDR3 was also found in at least 8 of 10 of them. CONCLUSIONS: The proposed method allows the ability to quickly identify the B-cell tumor-specific VH CDR3 using consensus primers while avoiding radioactive materials and bacterial cloning procedures

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G0/G1 phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3a/b and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34þ/CD4/CD7), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL

Induction of microRNAs, mir-155, mir-222, mir-424 and mir-503, promotes monocytic differentiation through combinatorial regulation

Acute myeloid leukemia (AML) involves a block in terminal differentiation of the myeloid lineage and uncontrolled proliferation of a progenitor state. Using phorbol myristate acetate (PMA), it is possible to overcome this block in THP-1 cells (an M5-AML containing the MLL-MLLT3 fusion), resulting in differentiation to an adherent monocytic phenotype. As part of FANTOM4, we used microarrays to identify 23 microRNAs that are regulated by PMA. We identify four PMA-induced micro- RNAs (mir-155, mir-222, mir-424 and mir-503) that when overexpressed cause cell-cycle arrest and partial differentiation and when used in combination induce additional changes not seen by any individual microRNA. We further characterize these prodifferentiative microRNAs and show that mir-155 and mir-222 induce G2 arrest and apoptosis, respectively. We find mir-424 and mir-503 are derived from a polycistronic precursor mir-424-503 that is under repression by the MLL-MLLT3 leukemogenic fusion. Both of these microRNAs directly target cell-cycle regulators and induce G1 cell-cycle arrest when overexpressed in THP-1. We also find that the pro-differentiative mir-424 and mir-503 downregulate the anti-differentiative mir-9 by targeting a site in its primary transcript. Our study highlights the combinatorial effects of multiple microRNAs within cellular systems.Comment: 45 pages 5 figure

Use of clofarabine for acute childhood leukemia.

A second-generation of purine nucleoside analogs, starting with clofarabine, has been developed in the course of the search for new therapeutic agents for acute childhood leukemia, especially for refractory or relapsed disease. Clofarabine is a hybrid of fludarabine and cladribine, and has shown to have antileukemic activity in acute lymphoblastic leukemia as well as in myeloid disorders. As the only new antileukemic chemotherapeutic agent to enter clinical use in the last 10 years, clofarabine was approved as an orphan drug with the primary indication of use in pediatric patients. Toxicity has been tolerable in a heavily pretreated patient population, and clofarabine has been demonstrated to be safe, both as a single agent and in combination therapies. Liver dysfunction has been the most frequently observed adverse event, but this is generally reversible. Numerous Phase I and II trials have recently been conducted, and are still ongoing in an effort to find the optimal role for clofarabine in various treatment strategies. Concomitant use of clofarabine, cytarabine, and etoposide was confirmed to be safe and effective in two independent trials. Based on the promising results when used as a salvage regimen, clofarabine is now being investigated for its potential to become part of frontline protocols

Use of clofarabine for acute childhood leukemia

A second-generation of purine nucleoside analogs, starting with clofarabine, has been developed in the course of the search for new therapeutic agents for acute childhood leukemia, especially for refractory or relapsed disease. Clofarabine is a hybrid of fludarabine and cladribine, and has shown to have antileukemic activity in acute lymphoblastic leukemia as well as in myeloid disorders. As the only new antileukemic chemotherapeutic agent to enter clinical use in the last 10 years, clofarabine was approved as an orphan drug with the primary indication of use in pediatric patients. Toxicity has been tolerable in a heavily pretreated patient population, and clofarabine has been demonstrated to be safe, both as a single agent and in combination therapies. Liver dysfunction has been the most frequently observed adverse event, but this is generally reversible. Numerous Phase I and II trials have recently been conducted, and are still ongoing in an effort to find the optimal role for clofarabine in various treatment strategies. Concomitant use of clofarabine, cytarabine, and etoposide was confirmed to be safe and effective in two independent trials. Based on the promising results when used as a salvage regimen, clofarabine is now being investigated for its potential to become part of frontline protocols

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications.

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4þ T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34þ/CD7/CD4) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL. Leukemia (2012) 26, 91–100; doi:10.1038/leu.2011.269; published online 4 October 201