Ewa Duszenko

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 ≥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.

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.

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)

Abstract 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+.

High incidence of ancestral HLA haplotype 8.1 and monoclonal incomplete DH–JH immunoglobulin heavy chain gene rearrangement in persistent polyclonal B-cell lymphocytosis

Dear Editor, Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare, benign, and usually asymptomatic entity, characterized by sustained B-cell CD5 lymphocytosis with normal surface ./1 chains ratio, with bilobulated and/or binuclear cells and elevated serum polyclonal IgM level. The increased frequency of HLA-DR7 haplotype and chromosomal clonal aberrations in particular concerning the chromosome 3 suggest a genetic background of this entity [1, 4]. We studied four women with PPBL, aged 48 to 51 years, for the clonality of rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in peripheral blood lymphocytes by 14 multiplex PCR reactions with different primers: three VH–JH (IGH), two DH–JH (IGH), two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), and one TCR delta (TCRD) [5]. Peripheral lymphocytes were also studied with classical cytogenetic and FISH analyses, using 3q/3pspecific probes. All patients and 200 healthy ethnically matched controls were HLA typed for A, B, Cw, and DRB1 genes by PCR-based sequence-specific primer amplification using SSP kits (Olerup, Sweden). Three out of four patients studied were asymptomatic, while one presented recurrent bacterial infections and decreased serum IgA and IgG levels. In all patients studied, peripheral lymphocytosis ranged from 6.1 to 10.5×10/l for a minimum of 5 years, with predominance of CD19/CD5 cells. Binucleated/ bilobulated lymphocytes were present on the blood smear, surface kappa/lambda chains ratios were normal, and serum levels of polyclonal IgM were increased. Cytogenetic studies revealed abnormalities in one patient (+i(3)(q10), +18, i(18)(q10), rob(13;14)(10;q10), with the presence of i(3)(q10) in 10% of interphase nuclei). No patients revealed the monoclonality in IGH VH–JH, IGK, IGL, nor TCR genes, but we found incomplete monoclonal DH7–JH IGH rearrangements in two patients. The observed DH–JH genes rearrangements were not related to other upstream DH or downstream JH genes. HLA typing showed DRB1*07 allele in 2/4 patients and in 58/200 controls (p=0.38). We found significantly higher frequency of Cw*07–B*08– Ann Hematol (2008) 87:597–598 DOI 10.1007/s00277-007-0434-z

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.

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.

Analiza genetycznych przyczyn niepowodzeń ciążowych u par z poronieniami i urodzeniem dziecka martwego lub żywego z wadami wrodzonymi

Wstep Czestośc wad u zywo urodzonych noworodkow wynosi od 0,94 do 4%, u martwo urodzonych jest kilkakrotnie wieksza i wynosi od ok. 9 do 20%. Spośrod wad o znanej etiologii ok. 35% jest spowodowanych przez czynniki wylącznie genetyczne. Cel pracy Celem pracy byla analiza genetycznych przyczyn niepowodzen ciązowych u par z poronieniami i urodzeniem dziecka martwego lub zywego z wadami wrodzonymi, z uwzglednieniem rodzaju wad plodow i noworodkow oraz kariotypow rodzicow. Material i metody Analize kariotypu rodzicow przeprowadzono na podstawie uzyskiwanych z 72 godzinnych hodowli limfocytow krwi obwodowej chromosomow metafazowych wybarwionych w technice GTG oraz, w pojedynczych przypadkach, w technice FISH. Wyniki Wśrod 59 par u 7 (11,85%) rozpoznano aberracje strukturalne. U 1 kobiety stwierdzono t(3;10)(p21;q21). Pozostale translokacje zrownowazone rozpoznano u mezczyzn: t(4;13)(q34;q31), t(10;22)(p11.2;q13), t(3;14)(p25;q24), t(13;18)(q14;q21.1). Ponadto u 1 kobiety rozpoznano translokacje robertsonowską; t(14;21)(q10;q10), a u innej niezidentyfikowaną aberracje chromosomu 16. Poznanie etiologii niepowodzen ciązowych pozwala na zwiekszenie efektywności zarowno poradnictwa genetycznego, jak i zapobiegania kolejnym niepowodzeniom.

Partial trisomy 4q and monosomy 9p in a girl with severe mental retardation, multiple anomalies and congenital hypoglycaemia

Unbalanced chromosomal aberrations are one of the most common causes of mental retardation and congenital defects. We analized genotype-phenotype correlations in a girl with dysmorphism, heart defect and hypoglycaemic attacks. The proband was born at term, after 2 years childless marriage, her birth weight was 2880 g. After birth, her phenotype was described as abnormal. She had microcephaly, scull with protruding sutures, narrow, short palpebral fissures, hypoplastic nose, hypoplastic nails, singular crease on right hand, clinodactyly of V fingers, excessive hair covering her face, shoulders and thighs, heart defect and vesico-uretheral reflux. She was also diagnosed as having hypothyroidism and hypoglycaemic attacks accompanied by seizures. Cytogenetic studies and FISH with WCP4 and WCP9 probes revealed an unbalanced karyotype: 46,XX,der(9)t(4;9)(q21;p22),9ph. The probands mothers karyotype was normal female with pericentric inversion 9. The probands father refused consent for cytogenetic studies.