Can Gene Methylation, Telomere Length and Activity of Telomerase Help with Individual Risk and Treatment Strategy of MDS Patients?

Background: Epigenetic de novo methylation of CpG-rich areas located in the promotors of gene p15^INK4b (CDKN2B) as it is capable to silence tumor suppressor genes and knowledge of telomere-telomerase complex may bring an important sign into molecular backround of malignant transformation in patients with myelodysplastic syndromes (MDS).

Aims: Intensity of gene methylation, the variability of telomere length (TRF) and telomerase activity (TA) were studied in bone marrow (BM) or peripheral blood (PB) cells of patients with MDS before treatment and during the course of the disease to evaluate its prognostic significance and association with clinical outcomes and disease progression.

Methods: 110 patients with MDS were stratiffied according to the WHO classification: 18 × RA, 3 × RARS, 32 × RCMD, 12 × 5q-syndrome, 17 × RAEB1, 19 × RAEB2 and 9 × MDS/MPS. BM cells from 30 age matched healthy donors served as controls, 5 leukemic cell lines were examined as positive controls. The level of DNA methylation was quantified by methylation specific PCR after sodium bisulphate modification of DNA, CpGenom DNA Modification kit from Scintila. The methylation status was dedicated as Methylation Indices (MI) in the range 0.0–1.0. The telomere length was determined as terminal restriction fragment (TRF index in kbp) after Southern analysis of genomic DNA, TeloTAGGG Telomere Length Assay kit from Roche. The activity of telomerase was assessed by Quantitative TRAP assay based on a photometric enzyme immunoassay on solid phase, Telo TAGGG Telomerase PCR Elisa plus kit from Roche. We postulated TRF shorter than 7.5 kbp as reduced telomeres and values of TA higher than 0.0263 as positive telomerase activity.

Results: Aberrant methylation of CDKN2B gene was present in 64% of MDS untreated patients showing significant heterogeneity in WHO subgroups. Mean values of MI increased as follows: from 0.18 ± 0.13 in RCMD, 0.21 ± 0.11 in RA/RARS, 0.26 ± 0.19 in 5q- syndrome, 0.33 ± 0.17 in RAEB1, 0.34 ± 0.17 in RAEB2, up to 0.41 ± 0.14 in MDS/MPS. A significant difference was found between early forms of MDS (RA/RARS, RCMD, 5q-syndrome) and advanced MDS with excess of blasts (RAEB1, RAEB2, MDS/MPS). High level of methylation CDKN2B gene was observed in 70% (33/47) of patients with advances forms of MDS (MI=0.36 ± 0.17) in comparison with early phases of MDS (MI=0.20 ± 0.14) in 60% (39/65) of patients. In one patient with RAEB2, a significant decrease in MI from 0.44 in BM and 0.37 in PB to 0,25 to 0,04, respectively was observed after 3 months of treatment with decitabine together with simultaneous prolongation of TRF from 7,62 kbp to 10.5 kbp. The patient achieved partial remission of the disease after 6 months of treatment with corresponding values of MI=0.20 and TRF=9.63kbp. In another patient, a progression of the disease from RCMD towards RAEB was connected vith increase in MI from 0.00 to 0.39 that correlated with elevation of telomerase activity from 0 to 0.0534.

Conclusions: The results confirm association of aberrant gene methylation with progression of early MDS to advanced forms with excess of blasts. Our findings also showed a certain degree of correlation between molecular changes in gene methylation, telomere length and telomerase activity in different phases of disease. Thus, determination of these markers may contribute to more precise estimation of an individual MDS patient’s risk and for decision of an optimal treatment strategy.