Whole-genome DNA methylation characteristics in pediatric precursor B cell acute lymphoblastic leukemia (BCP ALL).

In addition to genetic alterations, epigenetic abnormalities have been shown to underlie the pathogenesis of acute lymphoblastic leukemia (ALL)-the most common pediatric cancer. The purpose of this study was to characterize the whole genome DNA methylation profile in children with precursor B-cell ALL (BCP ALL) and to compare this profile with methylation observed in normal bone marrow samples. Additional efforts were made to correlate the observed methylation patterns with selected clinical features. We assessed DNA methylation from bone marrow samples obtained from 38 children with BCP ALL at the time of diagnosis along with 4 samples of normal bone marrow cells as controls using Infinium MethylationEPIC BeadChip Array. Patients were diagnosed and stratified into prognosis groups according to the BFM ALL IC 2009 protocol. The analysis of differentially methylated sites across the genome as well as promoter methylation profiles allowed clear separation of the leukemic and control samples into two clusters. 86.6% of the promoter-associated differentially methylated sites were hypermethylated in BCP ALL. Seven sites were found to correlate with the BFM ALL IC 2009 high risk group. Amongst these, one was located within the gene body of the MBP gene and another was within the promoter region- PSMF1 gene. Differentially methylated sites that were significantly related with subsets of patients with ETV6-RUNX1 fusion and hyperdiploidy. The analyzed translocations and change of genes' sequence context does not affect methylation and methylation seems not to be a mechanism for the regulation of expression of the resulting fusion genes.

Concomitance of monosomal karyotype with at least 5 chromosomal abnormalities is associated with dismal treatment outcome of AML patients with complex karyotype - retrospective analysis of Polish Adult Leukemia Group (PALG).

Monosomal karyotype (MK) and complex karyotype (CK) are poor prognostic factors in acute myeloid leukemia (AML). A comprehensive analysis of cytogenetic and clinical factors influencing an outcome of AML-CK+ was performed. The impact of cladribine containing induction on treatment results was also evaluated. We analyzed 125 patients with AML-CK+ treated within PALG protocols. MK was found in 75 (60%) individuals. The overall complete remission (CR) rate of 66 intensively treated patients was 62% vs. 28% in CK+ MK- and CK+ MK+ group (p = .01). No difference in CR rate was observed between DA and DAC arms. The overall survival (OS) in intensively treated patients was negatively influenced by MK, karyotype complexity (≥5 abnormalities), and WBC >20 G/L in multivariate analysis. The addition of cladribine to DA regimen improved OS only in MK- but not in MK+ group. In conclusion, concomitance of MK with ≥5 chromosomal abnormalities is associated with dismal treatment outcome in AMK-CK+.

CEBPA copy number variations in normal karyotype acute myeloid leukemia: Possible role of breakpoint-associated microhomology and chromatin status in CEBPA mutagenesis.

Copy number variations (CNV) in CEBPA locus represent heterogeneous group of mutations accompanying acute myeloid leukemia (AML). The aim of this study was to characterize different CEBPA mutation categories in regard to biological data like age, cytology, CD7, and molecular markers, and identify possible factors affecting their etiology. We report here the incidence of 12.6% of CEBPA mutants in the population of 262 normal karyotype AML (NK-AML) patients. We confirmed that double mutant AMLs presented uniform biological features when compared to single CEBPA mutations and accompanied mostly younger patients. We hypothesized that pathogenesis of distinct CEBPA mutation categories might be influenced by different factors. The detailed sequence analysis revealed frequent breakpoint-associated microhomologies of 2 to 12bp. The analysis of distribution of microhomology motifs along CEBPA gene showed that longer stretches of microhomology at the mutational junctions were relatively rare by chance which suggests their functional role in the CEBPA mutagenesis. Additionally, accurate quantification of CEBPA transcript levels showed that double CEBPA mutations correlated with high-level CEBPA expression, whereas single N-terminal CEBPA mutations were associated with low-level CEBPA expression. This might suggest that high-level CEBPA expression and/or accessibility of CEBPA locus contribute to B-ZIP in-frame duplications.

Different prognosis of acute myeloid leukemia harboring monosomal karyotype with total or partial monosomies determined by FISH: retrospective PALG study.

A monosomal karyotype (MK) was identified by banding techniques (BT) in acute myeloid leukemia (AML). However, BT may be insufficient to determine the actual loss of a complete chromosome, especially in complex karyotypes. We have investigated the effect of monosomy type, total (MK-t) and partial (MK-p), reevaluated by FISH, on prognosis. We have found that complete remission rate and probability of overall survival at 1 year was higher in MK-p (n=27) than MK-t (n=15) group (40% vs. 15.4%, P=0.19 and 30% vs. 9%, P=0.046, respectively). Our results indicate for the first time that monosomy type influences the prognosis of MK-AML.

Usefulness of classic cytogenetic testing compared to fluorescence in situ hybridization in genetic diagnosis of 58 patients with myelodysplastic syndromes.

INTRODUCTION: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal diseases of pluripotent hematopoietic stem or progenitor cells. MDS are characterized by ineffective hematopoiesis, increased apoptosis, peripheral blood cytopenias, and propensity to evolve into acute myelogenous leukemia. OBJECTIVES: The aim of our investigation was to compare the usefulness of classic cytogenetics and fluorescence in situ hybridization (FISH) to detect chromosome aberrations in myelodysplastic syndromes. PATIENTS AND METHODS: The study was carried out in a group of 58 patients with MDS. G-banding using trypsin and Giemsa (GTG banding) and FISH with a panel of five molecular probes for aberrations with prognostic significance in MDS (cen7/8, 5q31, 7q22/q35, 17p13, 20q13.3) were performed on bone marrow cells. RESULTS: The use of GTG technique allowed to detect chromosome aberrations in 25 (43.1%) subjects. However, the additional use of FISH showed the presence of aberrations also in additional 10 (17.2%) patients, which shifted 11 patients from one cytogenetic category to another. CONCLUSIONS: The use of FISH with MDS probe panel beside classic cytogenetics improves detection of chromosome aberrations, and also stratification of MDS patients to prognostic groups. Both methods should be used simultaneously in every genetically diagnosed MDS patient.

The application of conventional cytogenetics, FISH, and RT-PCR to detect genetic changes in 70 children with ALL.

We investigated bone marrow cells of 70 acute lymphoblastic leukemia children by conventional cytogenetics (CC), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR) methods. CC and RT-PCR for fusion genes BCR/ABL, MLL/AF4, E2A/PBX1, TEL/AML1 were performed at diagnosis in each patient. FISH was performed to verify the presence of fusion genes and MLL rearrangements and to estimate the percentage of abnormal cells. Karyotypes were obtained in 59 (84%) of 70 cases. Thirty-five (59%) of 59 cases revealed chromosome aberrations. Hyperdiploidy>50 chromosomes was present in nine cases, hyperdiploidy 47-50 chromosomes in six, pseudodiploidy in 15, and hypodiploidy in five. BCR/ABL was present in two cases, PBX1/E2A in two, and TEL/AML1 in 14. MLL/AF4 was not found, but the rearrangements of MLL gene were present in five children. The addition of RT-PCR and FISH to CC was of the utmost importance. One of two Ph translocations and one of two t(1;19) were first revealed by RT-PCR. Moreover, FISH showed the percentage of TEL/AML1(+) cells that turned to be an important prognostic factor. The outcome was the best for the children with hyperdiploidy>50 chromosomes without structural changes. It was also good for those with TEL/AML1 present in >or=80% of cells without chromosome aberrations. The presence of pseudodiploidy correlated with poor outcome. The outcome for patients with t(9;22)-BCR/ABL or 11q23-MLL rearrangement was the worst in study group. The presence of BCR/ABL caused eight times increase of risk of death; MLL rearrangements caused 12 times increase.

[The results of cytogenetic investigations in 107 couples with recurrent spontaneous abortions from Pomerania-Kujawy region of Poland]. / Wyniki badan cytogenetycznych u 107 par z regionu kujawsko-pomorskiego z nawracajacymi poronieniami samoistnymi.

About 10-15% of clinically diagnosed pregnancies end by spontaneous abortion. One of the causes of recurrent abortions is the presence of chromosome aberrations in a parent. The paper presents the results of cytogenetic investigations in 107 couples referred to genetic council clinic because of at least 2 spontaneous abortions. Cytogenetic analysis was performed on peripheral blood lymphocytes after standard 72h PHA-stimulated culture. At least 20 GTG- and CBG-banded metaphases were analyzed in each patient. Fluorescence in situ hybridization technique was used as to precisely define cytogenetic results. Chromosome aberrations were found in 7 couples (6.54%), exclusively in women. Numerical aberration (47,XXX) was present in 1 woman, and balanced structural aberrations in 6 (5.61%). In 3 of them balanced translocations were disclosed: t(7; 19)(p13;p13.3), t(8;16)(q24;q22), and t(3;8)(q21;p21), in 2--inversions: inv(2)(p25q31), inv(17)(p12p13.3), and in 1--der(20). Pericentric inversion of chromosome 9 was found in 3 men. The analysis of nongenetic factors showed that neither age, nor congenital anomalies of uterus could be an important factor causing abortions in analyzed couples with aberrations. However, infections and muta- or teratogenic exposure could contribute to loss of pregnancies in some cases. Authors conclude that karyotype analysis should be an integral part of diagnostics in couples with recurrent abortions.

[Cytogenetic changes and their prognostic significance in elderly acute myeloid leukemia]. / Zmiany cytogenetyczne i ich znaczenie rokownicze w ostrej bialaczce szpikowej u starszych osób.

Cytogenetic analyses were performed in 72 acute myeloid leukemia (AML) patients > or = 60 years old. Karyotype was normal in 35 (48.6%) patients (group III). 3 patients (4.2%) had favourable karyotype with t(15;17) as an isolated aberration (group IV). 21 patients (29.2%) had adverse karyotypes (group I) and 13 (18%) had intermediate karyotypes (group II). Adverse karyotypes were simple (< 3 aberrations), with add 3q, 5q-, 7q-, in 5 persons, and complex (> or = 3 aberrations) in 16. Karyotypes of 14 patients from the latter group contained > or = 5 aberrations. Laboratory and clinical data were comparable between groups with > or = 3 and with > or = 5 changes. In more than 2/3 complex karyotypes chromosome 5 and 7 aberrations also were found. AML clinical course of group II patients was more similar to that of group I than of groups III and IV. A frequency of complete remissions differed statistically between group I and the others and a frequency of complete and partial remissions together--between I + II and III + IV groups. Overall survival time differed statistically between all groups. There were significantly more patients with secondary AML in groups I and II than in group III. Analysis according to FAB did not show prognostic significance of this classification. Authors conclude that cytogenetics have a fundamental prognostic importance in AML of the elderly and should be taken into account in establishing therapeutic strategies.