Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML.

Assessment of the inflammatory process by endomyocardial biopsy in patients with dilated cardiomyopathy based on pathological and immunohistochemical methods.

INTRODUCTION: Myocarditis may lead to dilated cardiomyopathy (DCM) in immunogenetically predisposed individuals. The diagnosis of myocardial inflammation is currently based on histopathological and immunohistochemical methods. Previous studies indicate that inflammatory cardiomyopathy occurs in approximately 50% of patients with DCM. AIM: The goal of the study was to assess the inflammatory process in patients with DCM by endomyocardial biopsy using histopathological and immunohistochemical methods. METHODS: Endomyocardial biopsy specimens was examined using routine histopathological methods and immunochemical staining for T lymphocytes (CD3(+), n=84), major histocompatibility complex I (HLA ABC, n=48) and II (HLA DPQR, n=84) antigens and the adhesion molecules ICAM-1 (n=51) and VCAM-1 (n=48) in 84 patients (69 male, 15 female; mean age 35.0+/-10.5 years) with angiographically-confirmed DCM. Familial disease occurrence was noted in 14 (16.7%) patients. Cardiac samples obtained from 18 patients who died of non-cardiovascular causes were used as a control group. RESULTS: Myocarditis was diagnosed, according to the Dallas criteria, in 8 (9.5%) patients. The frequency of inflammatory cardiomyopathy, defined as the presence of >2 CD3(+) T lymphocytes per high-power field (hpf) in myocardial biopsy, was 14.3%. When broader criteria were applied (presence of >2.0 CD3(+) lymphocytes/hpf and/or 1.5 CD3(+) lymphocytes/hpf in multiple foci and increased expression of class I/II HLA), inflammatory cardiomyopathy was diagnosed in 32.1% of patients. Inflammatory activation of the endothelium, indicated by increased expression of at least three adhesion molecules (class I and II HLA, ICAM-1, VCAM-1), was present in 22 (45.8%) patients. The expression of HLA DPQR, HLA ABC and ICAM-1 was observed on the endothelium of capillaries and larger vessels, interstitial cells, and the surface of activated lymphocytes; immunohistochemical reactions were diffuse. In patients with markedly elevated expression of the aforementioned adhesion molecules, the expression was also present on cardiomyocyte cell membranes. VCAM-1 was restricted to the endothelium of individual small veins. The control group did not demonstrate any signs of myocarditis, inflammatory cardiomyopathy or inflammatory endothelial activation. CONCLUSIONS: The application of immunohistochemical methods to myocardial biopsy in order to identify the inflammatory cell phenotype and the presence of adhesion molecules permits the diagnosis of inflammatory cardiomyopathy in 14% or 32% of patients, depending on the criteria used, while conventional pathology allows for this diagnosis in 9% of patients. The observed frequency of inflammatory cardiomyopathy, defined as the presence of >2 CD3(+) T lymphocytes/hpf in the myocardium, was lower (14%) than in previous studies, while the frequency of inflammatory endothelial activation was similar (45%).