Diagnostic and Prognostic Power of Active DNA Demethylation Pathway Intermediates in Acute Myelogenous Leukemia and Myelodysplastic Syndromes

Abstract

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA. The active DNA demethylation process may be linked with aberrant methylation and can be involved in leukemogenesis. The levels of 5-methylcytosine oxidation products were analyzed in minimally invasive material: the cellular DNA from peripheral blood cells and urine of patients with AML and MDS along with the control group, using isotope-dilution two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. The receiver operating characteristic curve analysis was used for the assessment of the ability to discriminate patients’ groups from the control group, and AML from MDS. The most diagnostically useful for discriminating AML patients from the control group was the urinary excretion of 5-hydroxymethylcytosine (AUC = 0.918, sensitivity: 85%, and specificity: 97%), and 5-(hydroxymethyl)-2′-deoxyuridine (0.873, 74%, and 92%), while for MDS patients 5-(hydroxymethyl)-2′-deoxycytidine in DNA (0.905, 82%, and 98%) and urinary 5-hydroxymethylcytosine (0.746, 66%, and 92%). Multi-factor models of classification trees allowed the correct classification of patients with AML and MDS in 95.7% and 94.7% of cases. The highest prognostic value of the analyzed parameters in predicting the transformation of MDS into AML was observed for 5-carboxy-2′-deoxycytidine (0.823, 80%, and 97%) and 5-(hydroxymethyl)-2′-deoxyuridine (0.872, 100%, and 75%) in DNA. The presented research proves that the intermediates of the active DNA demethylation pathway determined in the completely non-invasive (urine) or minimally invasive (blood) material can be useful in supporting the diagnostic process of patients with MDS and AML. The possibility of an early identification of a group of MDS patients with an increased risk of transformation into AML is of particular importance.

Dynamic changes of DNA modifications: 5-hydroxymethyluracil and N6-methyladenine in the Drosophila melanogaster life cycle and in response to environmental factors

“…Our results suggest that a certain pool of 5-hmUra observed in D. melanogaster may arise from the enzymatic activity of dTET protein. What is interesting, the levels of 5-hmUra in the larvae and pupae DNA were signi cantly higher than these observed in human cells 41,42 , which suggests that that this modi cation plays an important role during the D. melanogaster development.…”

Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

“…Our results suggest that a certain pool of 5-hmUra observed in D. melanogaster may arise from the enzymatic activity of dTET protein. Interestingly, the levels of www.nature.com/scientificreports/ 5-hmUra in larval and pupal DNA were significantly higher than those observed in human cells 41,42 , which suggests that this modification plays an important role during D. melanogaster development. www.nature.com/scientificreports/ Interestingly, although D. melanogaster does not have L-gulonolactone oxidase (GULO, the enzyme responsible for the last stage of L-ascorbate synthesis), this vitamin plays an important role 43 .…”

The level of active DNA demethylation compounds in leukocytes and urine samples as potential epigenetic biomarkers in breast cancer patients

“…Leukocytes from heparinized blood samples were isolated using Histopaque 1119 solution (Merck) as per the manufacturer’s recommendations, following the storage in – 80 °C until analysis. DNA from leukocytes was isolated using the method described earlier 98 , 99 . See supplementary material .…”

“…Urine samples (2 mL) were spiked with a mixture of internal standards in 4:1 volumetric ratio. 2D-UPLC − MS/MS system consists of the gradient pump and autosampler for the first-dimension chromatography and the gradient pump and tandem quadrupole mass spectrometer with unispray ion source for the second-dimension chromatography 99 , 101 . See Supplementary information .…”