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.

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.

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+.

[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.

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.