Margit König

Incidence and relevance of secondary chromosome abnormalities in childhood TEL / AML1 + acute lymphoblastic leukemia: an interphase FISH analysis

The aim of the present study was to determine the frequency and clinical relevance of the most common secondary karyotype abnormalities in TEL/AML1+ B-cell precursor acute lymphoblastic leukemia (ALL) as assessed with fluorescence in situ hybridization (FISH) analyses. Screening of 372 patients who were enrolled in two consecutive Austrian childhood ALL multicenter trials identified 94 (25%) TEL/AML1+ cases. TEL deletions, trisomy 21 and an additional der(21)t(12;21) were detected in 52 (55%), 13 (14%) and 14 (15%) TEL/AML1+ patients, respectively. The 12p aberrations (P=0.001) and near tetraploidy (P=0.045) were more common in TEL/AML1+ patients, whereas the incidence of diploidy, pseudodiploidy, hypodiploidy, low hyperdiploidy, near triploidy, del(6q), chromosome 9 and 11q23 abnormalities was similar among TEL/AML1+ and TEL/AML1− patients. None of the TEL/AML1+ patients had a high hyperdiploid karyotype. Univariate analysis indicated that among TEL/AML1+ patients those with a deletion of the nontranslocated TEL allele had a worse prognosis than those without this abnormality (P=0.034). We concluded that the type and incidence of the most common secondary aberrations in TEL/AML1+ ALL can be conveniently identified with little additional effort during interphase screening with appropriate TEL and AML1 FISH probes. We also provided preliminary evidence that the deletion of the nontranslocated TEL allele may adversely influence the clinical course of TEL/AML1+ ALL.

PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13).

Recurrent chromosomal abnormalities present in malignant cells often define subentities with unique biological and clinical features. The molecular identification of genes involved in genetic alterations has led to the characterization of fusion genes with neoplastic properties. However, for many nonrandom translocations including the dic(9;12)(p11–13;p11–12), the molecular equivalent has not as yet been identified. The dicentric translocation dic(9;12) is a recurrent chromosome abnormality that accounts for close to 1% of childhood acute lymphoblastic leukemia (ALL). This specific alteration occurs almost exclusively in B-progenitor ALL, and unlike many other nonrandom translocations, is associated with an excellent prognosis. In this work, we provide strong evidence that the PAX5/ETV6 fusion transcript defines the clinical and biological entity that is associated with the presence of a dic(9;12) chromosome. As the PAX5 and ETV6 genes are localized at 9p13 and 12p13, respectively, the cytogenetic description of the dic(9;12)-PAX5/ETV6 rearrangement should be refined to dic(9;12)(p13;p13).

Comparison of p53 Mutational Status with mRNA and Protein Expression in a Panel of 24 Human Breast Carcinoma Cell Lines

We analyzed the p53 mutational status, mRNA and protein expression in 24 human breast carcinoma cell lines. Following measurement of their DNA content with flow cytometry, we ascertained the copy numbers of the centromere of chromosome 17 (cen17) and p53 with fluorescence in situ hybridization (FISH). A functional yeast assay (FASAY) was used to screen for inactivating mutations. Positive results were subsequently verified by DNA sequencing. Finally, we assessed the mRNA expression with a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay and the protein expression with immunocytochemical staining, western blot, and quantitative flow cytometry. The DNA content of the cell lines ranged from 0.85 to 2.58. Nine cell lines had concordant copy numbers (between two and four) of p53 and cen17, whereas 12 had more, and three less cen17 than p53 copies. The FASAY was successful in all but one cell line and revealed the presence of mutated alleles in 16 of them, 13 cell lines expressed only the mutated, and three both the mutated and the wild-type alleles. The mutations were comprised of 11 missense, two nonsense, and three frameshift mutations. Immunocytochemical staining, western blot and quantitative flow cytometry yielded comparable p53 protein expression results. However, both the mRNA and the protein expression levels varied considerably in the different cell lines and no consistent pattern with regard to the respective p53 mutational status became evident. The results obtained in these breast carcinoma cell lines indicate that no clear-cut linear relationship exists between the p53 mutational status and the extent of its respective mRNA and protein expression. Therefore, direct DNA analyses and functional assays remain the only methods for the reliable detection of p53 mutations.

Culture requirements for induction of dendritic cell differentiation in acute myeloid leukemia

Abstract Peripheral blood mononuclear cells (PBMCs) from 15 newly diagnosed acute myeloid leukemia (AML) patients were cultured in fetal calf serum-free media supplemented with either granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor α (TNFα), or GM-CSF, stem cell factor (SCF), TNFα and transforming growth factor β (TGFβ) in order to generate leukemia-derived dendritic cells (DCs). Cultured cells were analyzed by flow cytometry with respect to DC-associated surface molecules (CD1a, CD83, CD40, CD80, CD86, HLA-DR) when they showed significant DC morphology in culture (14 cases). After cultivation, neo-expression or upregulation of CD1a antigen was found in 8 samples, CD83 in 2, CD40 in 14, CD80 in 7, and CD86 in 9. Twelve of 14 AMLs, in which DC morphology could be induced upon cultivation, showed upregulation of at least 2 DC-associated molecules. For induction of DC differentiation, GM-CSF, IL-4 plus TNFα was superior in 11 cases, and better results were obtained with GM-CSF, SCF, TNFα plus TGFβ in 3 cases. In 7 of 14 samples tested, a marked increase of the T-cell stimulatory capacity could be demonstrated in the allogeneic mixed lymphocyte reaction. The leukemic origin of in vitro-generated DCs was demonstrated by fluorescence in situ hybridization in a patient with translocation t(15;17). Our results suggest that the use of different culture conditions may extend the number of AML patients in which a differentiation towards the DC lineage can be induced in vitro.

CBFB/MYH11 fusion in a patient with AML‐M4Eo and cytogenetically normal chromosomes 16

We present a unique case of acute myeloid leukemia M4Eo (AML‐M4Eo) with a CBFB/MYH11 fusion transcript and a trisomy 22, but in whom cytogenetic analyses did not disclose an inv(16). Fluorescence in situ hybridization (FISH) analysis with chromosome arm‐specific painting probes as well as with the c40 and c36 cosmids also revealed no evidence for an inv(16), whereas the application of locus‐specific probes confirmed the presence of a masked inv(16). The results of our comprehensive FISH investigations indicate that the events leading to this masked inv(16) were complex and concurred with deletions on both the long and short arms. The most likely explanation for the formation of the relevant CBFB/MYH11 fusion is an insertion of parts of the MYH11 into the CBFB gene, although it is also possible that it was formed by a double inversion.

Near-tetraploidy in childhood B-cell precursor acute lymphoblastic leukemia is a highly specific feature of ETV6/RUNX1-positive leukemic cases

Near‐tetraploidy (82–94 chromosomes) makes up fewer than 1% of childhood acute lymphoblastic leukemia (ALL) cases and has been reportedly associated with a possibly poorer prognosis compared with other ploidy groups. We analyzed 783 patients enrolled in the ALL‐BFM‐Austria 86, ‐90, ‐95, ‐99/2000 and Interfant‐Austria 99 trials in order to assess its incidence, biological characteristics, and prognostic relevance. Twelve of 783 patients (1.5%) had a near‐tetraploid ALL. Fluorescence in situ hybridization revealed that eight of the nine B‐cell precursor (BCP) cases and none of the three T‐cell ALL cases had an ETV6/RUNX1 rearrangement. After a median follow‐up of 11.4 years, none of the patients has relapsed or died. Thus, near‐tetraploidy appears to be a specific feature of ETV6/RUNX1+ BCP ALL cases that in turn may explain its excellent outcome.

EQUAL FREQUENCY OF TEL/AML1 REARRANGEMENTS IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA WITH AND WITHOUT DOWN SYNDROME

Constitutional trisomy 21 is the most prominent predisposing factor to childhood leukemia, whereas the t(12;21)(p13;q22) with its molecular genetic counterpart, the TEL/AML1 fusion gene, is the most common acquired chromosomal rearrangement in childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). Thus, it was somewhat surprising that according to the currently available literature the incidence of TEL/AML1+ BCP ALL is extremely low in patients with Down syndrome (DS). To further investigate this issue in a population-based fashion, the authors retrospectively assessed the number of DS patients with a TEL/AML1+ ALL in two consecutive Austrian ALL multicenter trials. Accordingly, they were able to analyze 8 of 10 individuals with DS and a BCP ALL, two of whom who suffered from a TEL/AML1+ leukemia. Based on this observation they concluded that individuals with BCP leukemia and a constitutional trisomy 21 may have similar likelihood to have a TEL/AML1 rearrangement as BCP ALL patients without this specific predisposing factor.

Generation of dendritic cells from human chronic myelomonocytic leukemia cells in fetal calf serum-free medium.

It is generally believed that the immune system plays a role not only in the acquisition of malignant diseases but also in the rejection of microscopic as well as established tumor cells. Failure of the immune system to eliminate tumor cells may be, among other factors, due to an insufficient presentation of tumor antigens. Dendritic cells (DCs), as professional antigen-presenting cells, therefore, may be therapeutically used to initiate or enhance immune responses in patients with malignancies. In this study we demonstrate that peripheral blood cells of patients with chronic myelomonocytic leukemia (CMML) can be induced to acquire DC characteristics. Upon culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) plus tumor necrosis factor α (TNFα), CMML cells develop DC morphology and acquire the phenotypic characteristics of DCs. When a CD14+ cell population is used for DC generation, a homogeneous differentiation towards the DC lineage occurs similar to that, observed in normal peripheral blood monocytes. CMML-derived DCs are potent stimulators of the allogeneic mixed lymphocyte reaction (MLR) when compared with uncultivated cells. The demonstration of a deletion of the long arm of chromosome 7, del(7)(q22), in 86% of highly enriched CD1a+ cells by fluorescence in situ hybridization (FISH) indicates the leukemic origin of generated DCs. In addition, we present data that generation of CMML-derived DCs is also possible under fetal calf serum-free conditions for a potential clinical use. These DCs may be used as a cellular vaccine to induce anti-tumor immunity in patients with CMML.

CD44 deficiency is a consistent finding in childhood Burkitt's lymphoma and leukemia

B-cell-derived non-Hodgkins lymphoma (NHL) of childhood and adolescence can be subdivided into mature B-cell neoplasms and B-cell precursor (BCP) lymphoblastic lymphomas (LBL). The former comprise Burkitts lymphoma and leukemia, diffuse large B-cell lymphoma (DLBCL) and primary mediastinal large B-cell lymphoma. With adequate therapy, about 80–90% of the patients with mature B-cell malignancies can now become long-term, disease-free survivors.1 However, the prognosis of children who suffer from a relapse is still very poor. In most therapy studies, risk stratification for mature B-cell NHL is largely based on stage of disease and the initial serum lactate dehydrogenase level.1 Moreover, to date, evaluation of minimal disseminated disease (MDD) at the time of diagnosis, that is in the bone marrow (BM) or peripheral blood, and of the kinetics of minimal residual disease (MRD) have not yet been implemented into the risk assignment of modern protocols. Nevertheless, as Burkitts lymphoma is genetically characterized by the presence of a chromosomal translocation involving the C-MYC oncogene on chromosome 8 and one of the loci of the immunoglobulin (Ig) heavy- or light-chain genes on chromosomes 14, 22 or 2, the molecular fusion products have been explored in a few polymerase chain reaction (PCR)-based studies to assess both minimal disseminated and residual diseases.2, 3 However, considering the long-time experience of different techniques on MRD analysis in childhood acute lymphoblastic leukemia (ALL), it seems very likely that besides PCR-based examinations of disease-specific fusion genes or clone-specific Ig gene rearrangements, flow cytometry could also allow MDD and MRD detection in Burkitts malignancies.4, 5

MRD levels during the first months of treatment indicate relapses in children with t(12;21)-positive ALL.

MRD levels during the first months of treatment indicate relapses in children with t(12;21)-positive ALL