Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene

<p>Abstract</p> <p>Background</p> <p>Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the <it>CDKN2B </it>(<it>p15</it>) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies of the myeloid lineage. Its promoter region usually shows heterogeneous methylation and is only rarely fully methylated. The methylation status of <it>CDKN2B </it>can be used as a biomarker of response to treatment. Therefore the accurate characterisation of its methylation is desirable.</p> <p>Results</p> <p>MS-HRM was used to assess <it>CDKN2B </it>methylation in acute myeloid leukaemia (AML) samples. All the AML samples that were methylated at the <it>CDKN2B </it>promoter (40/93) showed varying degrees of heterogeneous methylation. Six representative samples were selected for further study. dMS-HRM was used to simultaneously count the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM.</p> <p>Conclusion</p> <p>dMS-HRM is a powerful technique for the analysis of methylation in <it>CDKN2B </it>and other heterogeneously methylated genes. It eliminates both PCR and cloning bias towards either methylated or unmethylated DNA. Potentially complex information is simplified into a digital output, allowing counting of methylated and unmethylated alleles and providing an overall picture of methylation at the given locus. Downstream sequencing is minimised as dMS-HRM acts as a screen to select only methylated clones for further analysis.</p

The Development of Personalized Medicine: Acute Myeloid Leukemia as a Model

The term personalized medicine has been employed in widely different contexts and has acquired status as one of the most often used keywords recently. In this review we take it to understand the application of modern diagnostic medicine and therapeutics to patient with the purpose of eradicating disease or alleviating symptoms in a manner, where all actions are based on detailed knowledge of the condition of the individual patient. Applying these concepts should lead to optimization of clinical decision-making and, in its utmost consequence, a substantial decrease in costs incurred for hospitalization and follow-up. The latter is based on the evidence that for many disorders “less but more targeted” will mean improved outcome. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and is a major challenge in terms of diagnosis, care, follow-up and therapy. Thus, in population-based analyses, overall survival is only just exceeding 40% with major reasons for treatment failure. For these reasons, AML has been intensely studied during the recent decades. With the development of multiparametric flow cytometry, it allows us to get an accurate diagnosis and immunophenotypic profiles of AML. In addition, there is now an abundance of knowledge regarding its cytogenetic and molecular background. These enable us to follow the amount of disease down to the minutest quantity with a high resolution of molecular details. Finally, based on knowledge of these variables in the single patient cytoreduction is now being refined to therapies targeted to the molecular changes in the patient

Mutant CEBPA directly drives the expression of the targetable tumor-promoting factor CD73 in AML

The key myeloid transcription factor (TF), CEBPA, is frequently mutated in acute myeloid leukemia (AML), but the direct molecular effects of this leukemic driver mutation remain elusive. To investigate mutant AML, we performed microscale, in vivo chromatin immunoprecipitation sequencing and identified a set of aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. Comparing gene expression changes in human mutant AML and the corresponding mouse model, we identified , encoding CD73, as a cross-species AML gene with an upstream leukemic enhancer physically and functionally linked to the gene. Increased expression of CD73, mediated by the CEBPA-p30 isoform, sustained leukemic growth via the CD73/A2AR axis. Notably, targeting of this pathway enhanced survival of AML-transplanted mice. Our data thus indicate a first-in-class link between a cancer driver mutation in a TF and a druggable, direct transcriptional target

Cilostazol Inhibits Accumulation of Triglyceride in Aorta and Platelet Aggregation in Cholesterol-Fed Rabbits

Cilostazol is clinically used for the treatment of ischemic symptoms in patients with chronic peripheral arterial obstruction and for the secondary prevention of brain infarction. Recently, it has been reported that cilostazol has preventive effects on atherogenesis and decreased serum triglyceride in rodent models. There are, however, few reports on the evaluation of cilostazol using atherosclerotic rabbits, which have similar lipid metabolism to humans, and are used for investigating the lipid content in aorta and platelet aggregation under conditions of hyperlipidemia. Therefore, we evaluated the effect of cilostazol on the atherosclerosis and platelet aggregation in rabbits fed a normal diet or a cholesterol-containing diet supplemented with or without cilostazol. We evaluated the effects of cilostazol on the atherogenesis by measuring serum and aortic lipid content, and the lesion area after a 10-week treatment and the effect on platelet aggregation after 1- and 10-week treatment. From the lipid analyses, cilostazol significantly reduced the total cholesterol, triglyceride and phospholipids in serum, and moreover, the triglyceride content in the atherosclerotic aorta. Cilostazol significantly reduced the intimal atherosclerotic area. Platelet aggregation was enhanced in cholesterol-fed rabbits. Cilostazol significantly inhibited the platelet aggregation in rabbits fed both a normal diet and a high cholesterol diet. Cilostazol showed anti-atherosclerotic and anti-platelet effects in cholesterol-fed rabbits possibly due to the improvement of lipid metabolism and the attenuation of platelet activation. The results suggest that cilostazol is useful for prevention and treatment of atherothrombotic diseases with the lipid abnormalities