Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia.

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL.

Wnt signaling pathway is epigenetically regulated by methylation of Wnt antagonists in acute myeloid leukemia.

Activation of the Wnt signaling pathway has been implicated recently in the pathogenesis of leukemia. We studied the function of epigenetic regulation of the Wnt pathway and its prognostic relevance in acute myelogenous leukemia (AML). We used a methylation-specific polymerase chain reaction approach to analyze the promoter methylation status of a panel of Wnt antagonists including sFRP1, sFRP2, sFRP4, sFRP5, DKK1 and DKK3. Aberrant methylation of Wnt antagonists was detected in four AML cell lines and in up to 64% of AML marrow samples. Treatment of the cell lines with 5-aza-2'-deoxycytidine induced reexpression of methylated Wnt antagonists and inactivation of the Wnt pathway by downregulating the Wnt pathway genes cyclin D1, TCF1 and LEF1 and reducing nuclear localization of beta-catenin. In a subgroup of patients 60 years and younger with newly diagnosed AML and intermediate-risk cytogenetics, abnormal methylation of Wnt antagonists was associated with decreased 4-year relapse-free survival (28 vs 61%, respectively, P=0.03). Our results indicate a function of the epigenetic regulation of the Wnt pathway in predicting relapse in a subgroup of AML patients.

La enseñanza virtual de imágenes clínicas, tutorizadas mediante correo electrónico, es más eficiente que la enseñanza tradicional / On-line learning with e-mail tutoring for clinical images is more efficient than traditional teaching methods

Introducción. La enseñanza de la patología médica a través de imágenes es la clave para conseguir habilidad clínica en el diagnóstico. Objetivos. Comparar la eficacia docente de un método de enseñanza basado en imágenes en un entorno no presencial frente a la enseñanza tradicional en un entorno presencial, y evaluar el grado de satisfacción del alumno sobre este método de innovación docente en la enseñanza de la patología médica. Materiales y métodos. El estudio se realizó durante el curso 2005-2006 entre 62 alumnos de Patología médica, de la Facultad de Medicina de Córdoba. Se compararon los dos métodos de enseñanza: el presencial (en cada seminario se expusieron verbalmente 15 imágenes) y en el no presencial (imágenes clínicas expuestas en la página web de la Facultad de Medicina con tutorización a través del correo electrónico). La evaluación consistió en la valoración clínica de 16 imágenes (ocho imágenes de cada modelo de enseñanza). Se comparó la puntuación del examen teórico con la de las imágenes clínicas. Tras el examen final, el alumno realizó un cuestionario de evaluación. Resultados. La calificación media final de las imágenes (sobre 10 puntos) fue de: 6.8+/-1.5 y la del test (sobre 10 puntos) fue de 7.3+/-0.9. La calificación media de las imágenes expuestas en la web (7.4+/-1.6) fue superior a la del modelo presencial (6.3+/-1.7) p<0.0001. Existía una correlación entre la puntuación total de la imagen y la del test (r:0.584, p<0.0001). Asimismo, se correlacionaba la calificación de la imagen del modelo presencial y la e la imagen expuesta en al web (r:0.697, p<0.0001. Conclusiones. Las calificaciones de los casos expuestos en la web fueron superiores a las de los casos expuestos en los seminarios presenciales. La enseñanza de la patología médica a través de imágenes clínicas fue un método muy bien considerado por los alumnos (AU)

Introduction. The teaching of Internal Medicine through images is the key for the achievement of clinical skills in the diagnosis. Aims. To compare the efficiency of a teaching method based on images in a face-to-face learning situation with the traditional teaching methods, and to evaluate the degree of satisfaction amongst the alumni regarding the new teaching method within the Internal Medicine. Materials and methods. The study was carried out throughout the academic year 2005/06 amongst 62 Internal Medicine students in the School of Medicine in Córdoba. Two teaching methods were compared: (i) face-to-face- 15 images were exposed in workshop, and (ii)distance-clinical images were exposed at the School of Medicine web site through a tutorial system based on e-mail. The evaluation consisted in a clinical evaluation of 16 images-8 images for each teaching method. The results from the multiple choice exam and the clinical images exam were also compared. Finally, students answered an assessment questionnaire. Results. The final average qualification – out of 10- for the images was: 6.8+/-1.5 and fot the multiple choice exam – out of 10- was 7.3+/-0.9. The average qualification was higher for the images exposed at the web site than for the face-to-face model: (7.4+/-1.6) vs (6.3+/-1.7) p<0.0001. There was a correlation between the total punctuation for the images and the examination (r:0.584, p<0.0001). Moreover, there was also a correlation between the qualification obtained for the images in the face-to-face method and the images exposed on-line (r:0.697, p<0.0001). Conclusions: Qulaifications for the cases displayed on the website were higher than those for the cases exposed on the face-to-face workshops. In addition to this, students had a very positive opinion regarding the new teaching method for Internal Medicine based on clinical images (AU)

ASPP1, a common activator of TP53, is inactivated by aberrant methylation of its promoter in acute lymphoblastic leukemia.

We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.

Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/deletion polymorphisms.

Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 10(-4), as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3% of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01% (complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2% of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR.

Transcriptional silencing of the Dickkopfs-3 (Dkk-3) gene by CpG hypermethylation in acute lymphoblastic leukaemia.

DKK-3: is a newly characterised mortalisation-related gene and an antagonist of the Wnt oncogenic signalling pathway whose expression is decreased in a variety of cancer cell lines, suggesting that the Dkk-3 gene, located at chromosome 11p15.1, functions as a tumour suppressor gene. Although 11p15 is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Dkk-3 abnormalities has never been evaluated in this disease. We analysed CpG island methylation of the Dkk-3 promoter in six ALL cell lines and 183 ALL patients. We observed Dkk-3 hypermethylation in all cell lines and in cells from 33% (60/183) of ALL patients. Moreover, Dkk-3 methylation was associated with decreased Dkk-3 mRNA expression and this expression was restored after exposure to the demethylating agent 5-AzaC. Clinical features did not differ between hypermethylated and unmethylated patients. Estimated disease-free survival (DFS) and overall survival at 10 and 11 years, respectively, were 49.8 and 45.6% for normal patients and 10.5 and 15.1% for hypermethylated patients (P=0.001 and 0.09). Multivariate analysis demonstrated that Dkk-3 methylation was an independent prognostic factor predicting DFS (P=0.0009). Our data suggest that Dkk-3 methylation occurs at an early stage in ALL pathogenesis and probably influences the clinical behaviour of the disease.

Molecular heterogeneity in childhood precursor B acute lymphoblastic leukemia with immunoglobulin heavy chain gene in germline configuration.

Four childhood acute leukemias with morphological, cytochemical and immunological characteristics correlating to precursor B lymphocyte and with germline configuration of the immunoglobulin heavy chain joining region were studied for the organization of the C mu segment. Two of the four lymphoblast samples retained the germline configuration of both JH and C mu regions. The other two samples showed delection of the entire JH region resulting in the rearrangement of the C mu region. In contrast to patients with C mu rearrangement, patients with C mu in germline form were not able to achieve complete remission after induction therapy. Study of the C mu region in patients with JH segment in germline configuration could separate subgroups in childhood precursor B acute lymphoblastic leukemia with different prognoses.