Lipid Nanosystems Enhance the Bioavailability and the Therapeutic Efficacy of FTY720 in Acute Myeloid Leukemia.

Protein phosphatase 2A (PP2A) is a serin-threonin phosphatase that regulates many proteins critical for malignant cell behavior; therefore, PP2A is considered to be a human tumor suppressor. In this study, we assessed the pharmacokinetic profile and the antileukemic effects of the PP2A activator FTY720, free or encapsulated in lipid nanoparticles, in in vitro and in vivo models of acute myeloid leukemia. FTY720 lipid nanoparticles presented diameters around 210 nm, with an encapsulation efficiency up to 75% and significantly increased FTY720 oral bioavailability. In addition, FTY720 restores PP2A phosphatase activity and decreases phosphorylation of PP2A and its targets Akt, ERK1/2 and STAT5, all implicated in the pathogenesis of acute myeloid leukemia. Moreover, FTY720 exerts an additive anti-leukemic effect in combination with drugs used in standard induction therapy. Importantly, FTY720 lipid nanoparticles were more efficient at inducing cell growth arrest and apoptosis than FTY720 solution. Finally, oral administration of FTY720 lipid nanoparticles to mice every three days was as effective in reducing acute myeloid leukemia xenograft tumor growth as daily oral administration of FTY720. These results provide the first evidence for the potential use of FTY720 lipid nanoparticles as an oral therapeutic agent in acute myeloid leukemia.

Importance of genetics in acute myeloid leukemia.

Acute myeloid leukemia (AML) comprises a biologically and clinically heterogeneous group of aggressive disorders that occur as a consequence of a wide variety of genetic and epigenetic abnormalities in hematopoietic progenitors. Despite significant advances in the understanding of the biology of AML, most patients will die from relapsed disease. Whole-genome studies have identified novel recurrent gene mutations with prognostic impact in AML; furthermore, it is likely that in the near future genome-wide sequencing will become a routine for newly diagnosed patients with AML. Therefore, future clinical trials should aim to identify genetically defined high-risk patients, and further research is necessary to identify effective agents and develop new individualized therapeutic strategies for the treatment of this deadly disease.

Functional characterization of the promoter region of the human EVI1 gene in acute myeloid leukemia: RUNX1 and ELK1 directly regulate its transcription.

The EVI1 gene (3q26) codes for a transcription factor with important roles in normal hematopoiesis and leukemogenesis. High expression of EVI1 is a negative prognostic indicator of survival in acute myeloid leukemia (AML) irrespective of the presence of 3q26 rearrangements. However, the only known mechanisms that lead to EVI1 overexpression are 3q aberrations, and the MLL-ENL oncoprotein, which activates the transcription of EVI1 in hematopoietic stem cells. Our aim was to characterize the functional promoter region of EVI1, and to identify transcription factors involved in the regulation of this gene. Generation of seven truncated constructs and luciferase reporter assays allowed us to determine a 318-bp region as the minimal promoter region of EVI1. Site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays identified RUNX1 and ELK1 as putative transcription factors of EVI1. Furthermore, knockdown of RUNX1 and ELK1 led to EVI1 downregulation, and their overexpression to upregulation of EVI1. Interestingly, in a series of patient samples with AML at diagnosis, we found a significant positive correlation between EVI1 and RUNX1 at protein level. Moreover, we identified one of the roles of RUNX1 in the activation of EVI1 during megakaryocytic differentiation. EVI1 knockdown significantly inhibited the expression of megakaryocytic markers after treating K562 cells with TPA, as happens when knocking down RUNX1. In conclusion, we define the minimal promoter region of EVI1 and demonstrate that RUNX1 and ELK1, two proteins with essential functions in hematopoiesis, regulate EVI1 in AML. Furthermore, our results show that one of the mechanisms by which RUNX1 regulates the transcription of EVI1 is by acetylation of the histone H3 on its promoter region. This study opens new directions to further understand the mechanisms of EVI1 overexpressing leukemias.

Epigenetic regulation of miRNA genes in acute leukemia.

MicroRNAs (miRNAs) are small non-coding RNA molecules that can negatively regulate gene expression at the post-transcriptional level. miRNA expression patterns are regulated during development and differentiation of the hematopoietic system and have an important role in cell processes such as proliferation, apoptosis, differentiation or even in tumorigenesis of human tumors and in particular of hematological malignancies such as acute leukemias. Various miRNAs and their functions have been intensively studied in acute leukemias but the mechanisms that control their expression are largely unknown for the majority of aberrantly expressed miRNAs. miRNA expression can be regulated by the same genetic mechanism that modulate protein coding genes such as mutation, deletion, amplification, loss of heterozygosity and translocations. In this review we focus on the regulation of miRNAs in acute leukemias mediated by alterations in epigenetic mechanisms such as DNA methylation and histone code, describing the role of these alterations in the pathogenesis, diagnosis and prognosis of acute leukemias and their possible use as new therapeutic targets and biomarkers.

Desarrollo y conducta adaptativa en niños con insuficiencia renal crónica en DPCA / Development and adaptative behavior in children with chronic renal insuffiency on CAPD

Los niños con enfermedad renal crónica encuentran un riesgo aumentado de presentar compromiso de su neurodesarrollo. El objetivo de este trabajo fue la evaluación cognitiva y adaptativa de 15 niños con insuficiencia renal crónica en tratamiento de reemplazo con diálisis peritoneal ambulatoria. El desarrollo cognitivo fue normal o superior en 3 pacientes (20%) y retrasado en 12 (80%). La conducta adaptativa fue adecuada en 8 (53%) y baja o muy baja en 7 (47%). No hubo relación entre el desarrollo cognitivo y la escolaridad materna o el nivel socio económico, ni entre la conducta adaptativa y el nivel de escolaridad materna. La relación entre el NSE y la conducta adaptativa fue significativa (p 0.04). Esto sugeriría que si bien la enfermedad renal crónica compromete el desarrollo cognitivo, un nivel socioeconómico medio/alto puede proveer oportunidades para una mejor conducta adaptativa.

Children with chronic kidney disease are at increased riskof having impaired neurodevelopment. The objective of thepresent study was to perform a cognitive and adaptive eva-luation of 15 children with chronic renal insufficiency onoutpatient peritoneal dialysis. Cognitive development wasnormal or above normal in 3 patients (20%) and delayed in12 (80%). Adaptive behavior was adequate in 8 (53%) andlow or very low in 7 (47%). No correlation was found eitherbetween cognitive development and maternal education orsocio-economic level or between adaptive behavior andeducation of the mother. A significant correlation was foundbetween socio-economic level and adaptive behavior (p0.04). This would suggest that although chronic kidney dise-ase compromises cognitive development, middle or uppersocio-economic level may provide opportunities for impro-ved adaptive behavior.

PP2A impaired activity is a common event in acute myeloid leukemia and its activation by forskolin has a potent anti-leukemic effect.

Protein phosphatase 2A (PP2A) is a human tumor suppressor that inhibits cellular transformation by regulating the activity of several signaling proteins critical for malignant cell behavior. PP2A has been described as a potential therapeutic target in chronic myeloid leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia and B-cell chronic lymphocytic leukemia. Here, we show that PP2A inactivation is a recurrent event in acute myeloid leukemia (AML), and that restoration of PP2A phosphatase activity by treatment with forskolin in AML cells blocks proliferation, induces caspase-dependent apoptosis and affects AKT and ERK1/2 activity. Moreover, treatment with forskolin had an additive effect with Idarubicin and Ara-c, drugs used in standard induction therapy in AML patients. Analysis at protein level of the PP2A activation status in a series of patients with AML at diagnosis showed PP2A hyperphosphorylation in 78% of cases (29/37). In addition, we found that either deregulated expression of the endogenous PP2A inhibitors SET or CIP2A, overexpression of SETBP1, or downregulation of some PP2A subunits, might be contributing to PP2A inhibition in AML. In conclusion, our results show that PP2A inhibition is a common event in AML cells and that PP2A activators, such as forskolin or FTY720, could represent potential novel therapeutic targets in AML.

EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2.

BACKGROUND: The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans. METHODS: We performed QT-RT-PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT. RESULTS: Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines. CONCLUSIONS: Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

DNA profiling analysis of 100 consecutive de novo acute myeloid leukemia cases reveals patterns of genomic instability that affect all cytogenetic risk groups.

We have carried out a high-resolution whole genome DNA profiling analysis on 100 bone marrow samples from a consecutive series of de novo acute myeloid leukemia (AML) cases. After discarding copy number changes that are known to be genetic polymorphisms, we found that genomic aberrations (GA) in the form of gains or losses of genetic material were present in 74% of the samples, with a median of 2 GA per case (range 0-35). In addition to the cytogenetically detected aberration, GA were present in cases from all cytogenetic prognostic groups: 79% in the favorable group, 60% in the intermediate group (including 59% of cases with normal karyotype) and 83% in the adverse group. Five aberrant deleted regions were recurrently associated with cases with a highly aberrant genome (e.g., a 1.5 Mb deletion at 17q11.2 and a 750 kb deletion at 5q31.1). Different degrees of genomic instability showed a statistically significant impact on survival curves, even within the normal karyotype cases. This association was independent of other clinical and genetic parameters. Our study provides, for the first time, a detailed picture of the nature and frequency of DNA copy number aberrations in de novo AML.

Geographic differences in the incidence of cytogenetic abnormalities of acute myelogenous leukemia (AML) in Spain.

The incidence of chromosomal abnormalities in acute myeloid leukemia (AML) differs according to geographical regions in Spain. We analyse 1,271 consecutive patients diagnosed of AML between 1995 and 2002 in three different regions of Spain: northern, central and southern. There were 624 males (55%) and 505 females (45%). Age ranged between 1 month and 94 years with a median of 61 years. Abnormal karyotypes were observed in 64% of cases. Numerical abnormalities as sole cytogenetic changes were detected in 15% of patients, while structural aberrations were present in 28% of cases, and both abnormalities were found in 22% of patients. A significantly higher proportion of t(15;17) was observed in the south of Spain (21.6%) than in the central (17%) or northern regions (12.6%) (p=0.03). By contrast, patients from the south of Spain showed lower incidence of t(8;21) (0%, compared to 1.6% and 3.6% in central and northern areas, respectively, p=0.04). These differences were maintained in the age-adjusted analysis. Trisomy 8 showed similar incidence in southern and central areas, while the incidence in the northern area was lower (14% and 10%, respectively, p=0.04). Other chromosomal abnormalities, such as inv(16) or 11q23 rearrangements, were found at similar frequencies in the three regions.

Disruption and aberrant expression of HMGA2 as a consequence of diverse chromosomal translocations in myeloid malignancies.

Chromosomal translocations that target HMGA2 at chromosome band 12q14 are seen in a variety of malignancies, notably lipoma, pleomorphic salivary adenoma and uterine leiomyoma. Although some HMGA2 fusion genes have been reported, several lines of evidence suggest that the critical pathogenic event is the expression of truncated HMGA2 isoforms. We report here the involvement of HMGA2 in six patients with myeloid neoplasia, dysplastic features and translocations or an inversion involving chromosome bands 12q13-15 and either 7p12, 8q22, 11q23, 12p11, 14q31 or 20q11. Breaks within or very close to HMGA2 were found in all six cases by molecular cytogenetic analysis, leading to overexpression of this gene as assessed by RT-PCR. Truncated transcripts consisting of HMGA2 exons 1-2 or exons 1-3 spliced to intron-derived sequences were identified in two patients, but were not seen in controls. These findings suggest that abnormalities of HMGA2 play an important and previously unsuspected role in myelodysplasia.

Abnormalities on 1q and 7q are associated with poor outcome in sporadic Burkitt's lymphoma. A cytogenetic and comparative genomic hybridization study.

Comparative genomic hybridization (CGH) studies have demonstrated a high incidence of chromosomal imbalances in non-Hodgkin's lymphoma. However, the information on the genomic imbalances in Burkitt's Lymphoma (BL) is scanty. Conventional cytogenetics was performed in 34 cases, and long-distance PCR for t(8;14) was performed in 18 cases. A total of 170 changes were present with a median of four changes per case (range 1-22). Gains of chromosomal material (143) were more frequent than amplifications (5) or losses (22). The most frequent aberrations were gains on chromosomes 12q (26%), Xq (22%), 22q (20%), 20q (17%) and 9q (15%). Losses predominantly involved chromosomes 13q (17%) and 4q (9%). High-level amplifications were present in the regions 1q23-31 (three cases), 6p12-p25 and 8p22-p23. Upon comparing BL vs Burkitt's cell leukemia (BCL), the latter had more changes (mean 4.3 +/- 2.2) than BL (mean 2.7 +/- 3.2). In addition, BCL cases showed more frequently gains on 8q, 9q, 14q, 20q, and 20q, 9q, 8q and 14q, as well as losses on 13q and 4q. Concerning outcome, the presence of abnormalities on 1q (ascertained either by cytogenetics or by CGH), and imbalances on 7q (P=0.01) were associated with a short survival.

Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas.

AIMS: The aim of this work is the study of the prognostic significance of the chromosomal aberrations described in a series of invasive ductal breast carcinomas. METHODS AND RESULTS: We analysed by comparative genomic hybridization a group of 70 formalin-fixed paraffin-embedded invasive ductal breast carcinomas. Aberrations showed a frequency similar to previous studies using frozen tumours. Interestingly, we identified gains involving 6q16-q24 more frequently than in other series. We analysed the association among the chromosomal imbalances, 11 histopathological factors, relapse rate and overall survival of patients. Associations showed 16q losses as a potential marker of good prognosis, as they were more frequent in node-negative (P=0.025) and in oestrogen-positive tumours (P < 0.001). Furthermore, 100% of bcl-2+ tumours presented this aberration compared with 29.3% in bcl-2- (P=0.014). 1q, 11q, 17q and 20q gains were associated with poor prognosis: 95% of cases with 1q gains were bigger than 20 mm (P=0.041). Tumours with 1q and 11q gains showed a higher relapse rate (P=0.063; P=0.066). Within the good prognosis group of lymph node-negative patients, 17q and 20q gains identify a subgroup with increased relapse rate (P=0.039). CONCLUSIONS: Chromosomal imbalances, together with histopathological factors, may help to predict outcome in breast cancer patients.

Use of malaria prevention measures by North American and European travelers to East Africa.

BACKGROUND: The use of preventive measures, including effective chemoprophylaxis, is essential for protection against malaria among travelers. However, data have shown that travelers and medical advisors are confused by the lack of uniform recommendations and numerous prophylactic regimens of varying effectiveness that are used. METHODS: To assess the use and type of preventive measures against malaria, we conducted a cross-sectional study in 1997 among travelers departing from the Nairobi and Mombasa airports in Kenya with European destinations. RESULTS: Seventy-five percent of the travelers studied were residents of Europe and 25% were residents of North America; all stayed less than 1 year, and visited malarious areas. Most travelers, 97.1%, were aware of the risk and 91.3% sought pretravel medical advice. Although 95.4% used chemoprophylaxis and/or antimosquito measures, only 61.7% used both regular chemoprophylaxis and two or more antimosquito measures. Compliance with chemoprophylaxis was lowest amongst those who used a drug with a daily, as opposed to, a weekly dosing schedule, stayed more than 1 month, attributed an adverse health event to the chemoprophylaxis, and were less than 40 years of age. Among US travelers, 94.6% of those taking chemoprophylaxis were taking an effective regimen, that is, mefloquine or doxycycline. Only 1.9% used a suboptimal drug regimen, such as chloroquine/proguanil. Among European travelers, 69% used mefloquine or doxycycline, and 25% used chloroquine/proguanil. Notably, 45.3% of travelers from the UK used chloroquine/proguanil. Adverse events were noted by 19.7% of mefloquine users and 16.4% of travelers taking chloroquine/proguanil. Neuropsychologic adverse events were reported by 7.8% of users of mefloquine and 1.9% of those taking chloroquine/proguanil. The adverse events, however, had a lesser impact on compliance than frequent dosing schedule. CONCLUSIONS: Health information should be targeted to travelers who are likely to use suboptimal chemoprophylaxis or may be noncompliant with prophylaxis. Uniform recommendations for effective chemoprophylaxis with simple dosing schedules are necessary to reduce rates of malaria among travelers to Africa.

Comparative genomic hybridization and amplotyping by arbitrarily primed PCR in stage A B-CLL.

Cytogenetic analysis is useful in the diagnosis and to assess prognosis of B-cell chronic lymphocytic leukemia (B-CLL). However, successful cytogenetics by standard techniques has been hindered by the low in vitro mitotic activity of the malignant B-cell population. Fluorescence in situ hybridization (FISH) has become a useful tool, but it does not provide an overall view of the aberrations. To overcome this hurdle, two DNA-based techniques have been tested in the present study: comparative genomic hybridization (CGH) and amplotyping by arbitrarily primed PCR (AP-PCR). Comparative genomic hybridization resolution depends upon the 400-bands of the human standard karyotype. AP-PCR allows detection of allelic losses and gains in tumor cells by PCR fingerprinting, thus its resolution is at the molecular level. Both techniques were performed in 23 patients with stage A B-CLL at diagnosis. The results were compared with FISH. The sensitivity of AP-PCR was greater than CGH (62% vs. 43%). The use of CGH combined with AP-PCR allowed to detect genetic abnormalities in 79% (15/19) of patients in whom G-banding was not informative, providing a global view of the aberrations in a sole experiment. This study shows that combining these two methods with FISH, makes possible a more precise genetic characterization of patients with B-CLL.