Epigenetic de novo methylation of CpG islands is an important event in malignant transformation. Two genes are frequently methylated: cyclin-dependent kinase inhibitor 2B (CDKN2B) and cyclin-dependent kinase inhibitor 2A (CDKN2A). In our study methylation of these genes was studied in 63 patients with myelodysplastic syndromes (MDS), 2 with myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and 13 with acute myeloid leukemia (AML). Five patients were monitored during 5-azacytidine treatment. Twenty-six healthy donors were tested in a control group. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) method with all associated techniques was used for detection. Aberrant methylation was present in the CDKN2A gene in 38% and in the CDKN2B gene in 77% of the patients in MDS group. The level of methylation was higher in the group of AML patients - 77% in CDKN2A gene and 100% in CDKN2B gene. In MDS patients, an aberrant methylation was associated with a tendency to disease progression towards more advanced forms according to the World Health Organization (WHO) classification and the International Prognostic Scoring System (IPSS). Significant differences in methylation level were observed between early and advanced forms of MDS in CDKN2B gene (P value < 0.05) but not for CDKN2A gene. The trend of methylation in patients treated with azacitidine was analyzed in CDKN2B gene and correlated with the course of the disease. Increased methylation was connected with disease progression. We concluded that the methylation level of CDKN2B gene might be used as a marker of leukemic transformation in MDS. Our study indicates the role of hypermethylation as an important event in the progression of MDS to AML.
Diffuse astrocytomas and oligodendrogliomas (WHO grade II) are the most common histological subtypes of low-grade gliomas (LGGs). Several molecular and epigenetic markers have been identified that predict tumor progression. Our aim was in detail to investigate the genetic and epigenetic background of LGGs and to identify new markers that might play a role in tumor behavior. Twenty-three patients with oligodendroglioma or oligoastrocytoma (LGO) and 22 patients with diffuse astrocytoma (LGA) were investigated using several molecular-cytogenetic and molecular methods to assess their copy number variations, mutational status and level of promoter methylation. The most frequent findings were a 1p/19q codeletion in 83% of LGO and copy-neutral loss of heterozygosity (CN-LOH) of 17p in 72% of LGA. Somatic mutations in the isocitrate dehydrogenase 1 or 2 (IDH1/IDH2) genes were detected in 96% of LGO and 91% of LGA. The O-6-methylguanine-DNA-methyltransferase (MGMT) promoter was methylated in 83% of LGO and 59% of LGA. MutL homolog 3 (MLH3) promoter methylation was observed in 61% of LGO and 27% of LGA. Methylation of the MGMT promoter, 1p/19q codeletion, mutated IDH1, and CN-LOH of 17p were the most frequent genetic aberrations in LGGs. The findings were more diverse in LGA than in LGO. To the best of our knowledge, this is the first time description of methylation of the MLH3 gene promoter in LGGs. Further studies are required to determine the role of the methylated MLH3 promoter and the other aberrations detected.
Overexpressed Wilms tumor gene 1 (WT1) has been found in a majority of patients with acute myeloid leukemia (AML). The aim of this study was to confirm the applicability of WT1 expression measurement as a marker of minimal residual disease (MRD). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood (PB) according to European Leukemia Net (ELN) recommendations. The WT1 expression was related to the expression of a reference gene Abelson (ABL) and the results were calculated as a number of WT1 copies related to 104 copies of ABL gene. The upper normal limit of WT1 expression was set at 50 copies of WT1 to 104 copies of ABL. Morphological, flow cytometry and chimerism examinations were evaluated according to standard protocols.A total of 51 AML patients with overexpressed WT1 gene were analyzed. The median follow-up after transplantation was 14 (2-72) months. WT1 expression levels exceeding the upper normal limit were considered as a sign of impending hematological relapse, in accord with morphological, flow cytometry and chimerism data, as well as with the expression of the specific fusion genes. Moreover, in 7 patients the rise of WT1 expression preceded all other standard methods. Patients with high WT1 expression before allogeneic hematopoietic stem cell transplantation (allo-HSCT) had significantly worse outcome than patients with low WT1 level. Examination of WT1 expression in PB of patients with AML is a useful tool for MRD monitoring. Moreover, the WT1 gene expression before stem cell transplantation seems to be of prognostic significance.
Telomere length was evaluated by terminal repeat fragment method in 66 previously untreated patients with B-chronic lymphocytic leukemia (B-CLL) to ascertain whether telomere shortening was associated with genomic aberrations, immunoglobulin variable heavy chain (IgVH) mutational status, CD38 and ZAP-70 expression, and telomerase activity. Chromosomal aberrations were present in peripheral blood cells of 73% patients (48/66), no difference in telomere length between patients with good and intermediate prognosis according to cytogenetics was found. Association between telomere length and IgVH mutational status, ZAP-70 and CD38 expression was proved as significantly shorter telomeres in patients with unmutated IgVH status (p=0.01) and ZAP-70 positivity (p=0.01) and CD38 positivity (p=0.05) were detected. Telomerase activity was positive in 11 patients out of 21 examined, correlation between telomere length and telomerase activity was found (p=0.05). Telomere length and telomerase activity in combination with other prognostic parameters complete the risk profile of B-CLL patients and might serve for an easy decision on optimal treatment strategy.
Cell chimerism determination is important for the monitoring of engraftment dynamics and for relapse prediction. Our cohort of 474 patients was divided into two groups according to the determination methods used over time, and by their chimerism status. A significant difference in survival was observed between mixed vs complete chimerism (P < 0.0001 vs P < 0.0002) in both patient groups, and also vs microchimerism (P = 0.0201) in the second group. Detection of mixed chimerism is thus a high‐risk factor, and microchimerism is potentially a risk factor in the post‐transplantation course. Methods with a high sensitivity for monitoring cell chimerism significantly improve the assessment of patients post‐transplant, and they enable the identification of patients with high relapse risk. Supported by MH CZ—DRO (00023736, UHKT).
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