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.

Etoposide-initiated MLL rearrangements detected at high frequency in human primitive hematopoietic stem cells with in vitro and in vivo long-term repopulating potential.

Rearrangements initiating within the well-characterized break-point cluster region of the mixed lineage leukemia (MLL) gene on 11q23 are a hallmark of therapy-related leukemias following treatment with topoisomerase II poisons including etoposide. Hematopoietic stem cells (HSC) are believed to be the target cell for leukemia-initiating MLL rearrangement events. Although etoposide treatment is sufficient to induce readily detectable MLL rearrangements in primary human CD34+ cells, the majority of cells that gain translocations do not proliferate in culture possibly due to reduced proliferative capacity of most CD34+ cells during normal differentiation [Blood 2005;105:2124]. We characterized the impact of etoposide on primary human long-term repopulating HSC that represent only a minor portion of CD34+ cells. The proliferative capacity of HSC is dramatically increased following both a single and multiple exposures to etoposide as determined by their ability to engraft bone marrow of immune-deficient non-obese diabetic/severe combined immunodeficient mice and to initiate hematopoiesis in long-term initiating cultures. Similar to results in CD34+ cells, a significant proportion of etoposide-treated HSC-derived clones harbored stable MLL rearrangements, including duplications, inversions and translocations. These results indicate HSC are highly susceptible to etoposide-induced and potentially oncogenic rearrangements initiating within MLL, and these HSC are particularly proficient for continued long-term proliferation both in vivo and in vitro.

Association of TNFd and IL-10 polymorphisms with mortality in unrelated hematopoietic stem cell transplantation.

BACKGROUND: Non-HLA immunogenetic polymorphisms may influence outcome of hematopoietic stem cell transplantation (HSCT). In this study, we have determined the role of TNFa, TNFd, IL-10, IL-1, IL-1Ra, and IL-4R polymorphisms in patients transplanted with HSC of an unrelated donor. METHODS: The allelic variants of four SNPs (IL-10-1082, IL-1beta-511, IL-4R-3223, IL-4R-1902) and four microsatellites (TNFa, TNFd, IL-10-1064, IL-1Ra) were determined in 131 unrelated patient/donor pairs typed for HLA-A/B/C/DR/DQ (four digits). RESULTS: The allelic distribution of the polymorphisms was similar to that previously reported in Caucasoid populations. Patient and donor TNFd and patient IL-10-1064 polymorphisms correlated with mortality in univariate analysis. Patients with TNFd1/d2/d3 genotypes had 3-year survival rates of 65%. A gradual decrease in survival rates was observed for patients with TNFd3/d3 genotypes (50%, p=n.s.), TNFd4 (46%, P=0.08), and TNFd5 (33%, P=0.03). A multivariate analysis of 10/10 matched patients revealed that the following patient genotypes correlated with lower survival: TNFd3/d3 (RR 4.08, P=0.026) TNFd4 (RR 3.78, P=0.032) and TNFd5 (RR 6.69, P=0.021) all compared to TNFd1/d2/d3 genotypes. Patient IL-10 (12, 14, 15) microsatellite alleles correlated with lower 3-year survival (28%) when compared to IL-10 (<12) (56%, P=0.052) and to Il-10 (13) alleles (60%, P=0.0023). In multivariate analysis this correlation remained significant only in recipients of HSCT of 10/10 HLA matched donors (RR=2.96, P=0.038). CONCLUSION: The data demonstrate a significant correlation of the TNFd and IL-10-1064 microsatellite polymorphisms with mortality after unrelated HSCT. They support the hypothesis that simple genomic tests, in addition to precise HLA matching, may contribute to determine prognosis in patients undergoing unrelated HSCT.

Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion.

Rearrangements involving the MLL gene on chromosome band 11q23 are a hallmark of therapy-related acute myeloid leukemias following treatment with topoisomerase II poisons including etoposide. Therapy-related and de novo genomic translocation breakpoints cluster within a well-characterized 8.3-kb fragment of MLL. Repair of etoposide-stabilized DNA topoisomerase II covalent complexes may initiate MLL rearrangements observed in patients. We used a culture system of primary human hematopoietic CD34+ cells and inverse polymerase chain reaction to characterize the spectrum of stable genomic rearrangements promoted by etoposide exposure originating within an MLL translocation hotspot in therapy-related leukemia. Alterations to the region were observed at a readily detectable frequency in etoposide-treated cells. Illegitimate repair events after minimal repair included MLL tandem duplications and translocations, with minor populations of deletions or insertions. In stably repaired cells that proliferated for 10 to 14 days, the significant majority of illegitimate events were MLL tandem duplications, and several deletions, inversions, insertions, and translocations. Thus, etoposide promotes specific rearrangements of MLL consistent with the full spectrum of oncogenic events identified in leukemic samples. Although etoposide-initiated rearrangements are frequent, only a small subset of translocations occurs in cells that proliferate significantly.

CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion.

We hypothesized that the CXC chemokine receptor-4 (CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4(+) tumor cells into the bone marrow and lymph nodes, which secrete the alpha-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3- and PAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHR gene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4-SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1) phosphorylation of mitogen-activated protein kinase p42/44; (2) locomotion; (3) directional chemotaxis across membranes covered by laminin, fibronectin, or Matrigel; (4) adhesion to laminin, fibronectin, and endothelial cells; and (5) increased MMP-2 and diminished tissue inhibitors of metalloproteinases secretion. The small-molecule CXCR4-specific inhibitor, T140, effectively blocked the in vitro responses of RMS cells to SDF-1. On the basis of these observations we suggest that the CXCR4-SDF-1 axis may play an important role in tumor spread and metastasis of RMS cells to bone marrow and that molecular strategies aimed at inhibiting this axis could thus prove to be useful therapeutic measures.

Polymorphic tumor necrosis factors microsatellite TNFa4 is associated with resistance of Hodgkin lymphoma to chemotherapy and with replapses after therapy.

BACKGROUND: The response of tumors to chemotherapy (CHT) exhibits wide individual variations. PATIENTS AND METHODS: We examined the incidence of polymorphic TNF genes in 61 patients treated for Hodgkin lymphoma. RESULTS: During treatment, the patients were divided as responders or non-responders, depending upon the amount of CHT required for a clinical eradication of the tumor. The incidence of TNFa4, a microsatellite allele associated with low TNF production in leukocytes, was significantly higher in responders than in non-responders (25.7% vs 0 %, p=0.04). We also examined the incidence of tumor relapses 2-5 years after treatment. The incidence of TNFa4 was also significantly higher in patients with relapses, than in those without relapses (41.1% vs 9.3%, p=0.007). CONCLUSION: These results indicate that TNFa4 is a marker of resistance of Hodgkin lymphoma to chemotherapy and most probably is a marker of bad prognosis.