Purpose We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias. Experimental Design ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols. Results We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup. Conclusions Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.

Approximately 25% of childhood B-cell precursor acute lymphoblastic leukemia have an ETV6/RUNX1 (E/R) gene fusion that results from a t(12;21). This genetic subgroup of leukemia is associated with near-triploidy, near-tetraploidy, and trisomy 21 as rather specific types of secondary changes. Here, we show that, unlike various controls, E/R-expressing Ba/F3 clones acquire a tetraploid karyotype on prolonged culture, corroborating the assumption that E/R may attenuate the mitotic checkpoint (MC). Consistent with this notion, E/Rexpressing diploid murine and human cell lines have decreased proportions of cells with 4N DNA content and a lower mitotic index when treated with spindle toxins. Moreover, both RUNX1 and E/R regulate mitotic arrestdeficient 2 L1 (MAD2L1), an essential MC component, by binding to promoter-inherent RUNX1 sites, which results in down-regulation of MAD2L1 mRNA and protein in E/R-expressing cells. Forced expression of E/R also abolishes RUNX1-induced reporter activation, whereas E/R with a mutant DNA-binding site leads to only minor effects. Our data link for the first time E/R, MC, and MAD2L1 and provide new insights into the function of the E/R fusion gene product. Although tetraploidy is an almost exclusive feature of E/R-positive leukemias, its rarity within this particular subgroup implies that further yet unknown factors are required for its manifestation.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonalityFafter correction for the respective copy number of IGH allelesFwas only 16%. Monoclonal and oligoclonal HeH cases had predominantly V H to preexisting DJ H recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which V H replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.

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B cell precursor (BCP) ALL is usually a monoclonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, a clone with disomy 14 can have a maximum of two unique rearrangements. In contrast, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor either a maximum of three unique or two unique rearrangements together with a third that may share particular sequences with the one or the other The pattern of IGH rearrangements in cases with trisomy 14 may therefore be misinterpreted to be oligoclonal if the chromosome 14 copy number is not known. Since oligoclonal IGH rearrangements may be instable at relapse, they generally are not used for minimal residual disease (MRD) analysis. Thus, in HeH patients seemingly oligoclonal IGH rearrangements may undeserved be skipped as MRD target. We investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 consecutively recruited HeH BCP ALL. This cohort was used for assessing overall frequencies. To enrich the number of small subgroups, 40 selected HeH cases were added. Cytogenetic and FISH analyses were performed according to standard procedures. IGH rearrangements were determined according to standardized ESG-MRD protocols. Even though the majority of HeH cases (78/90, 87%) had an extra chromosome 14, there was a small but distinct subgroup comprising 13% (12/90) of HeH cases with a disomy 14. Overall, IGH rearrangements were present in about 95% of leukemias representing incomplete DJH rearrangements in about 40% of cases. More than two IGH rearrangements and/or related rearrangements were found in 44% of the same HeH cohort with an overall frequency of 16% “true” oligoclonality after correction for the actual number of chromosomes 14. Of note, leukemias with only two copies of chromosome 14 revealed a significantly higher frequency of apparent oligoclonality compared to those with three copies of chromosome 14 (36% versus 13%). Monoclonal HeH leukemias with trisomy 14 could neither be distinguished from their oligoclonal counterparts nor from oligoclonal TEL-AML1 positive and “not further genetically discriminated” BCP ALLs based on the number of IGH rearrangements per cases and the type of secondary rearrangements (VH or DH to DJH or VH replacement). However, the patterns of secondary rearrangements had shifted from a predominantly VH to DJH recombination in the former towards VH replacement in the latter two groups. Our data have implications for MRD analysis, since oligoclonal patterns of IGH rearrangements account for about 25–30% of childhood BCP ALL. Hence, the interpretation of whether a particular IGH rearrangement pattern is really clonal or not may be crucial in some of these cases and may be better defined by taking into account at least the genetic subtype of the respective leukemia (i.e. hyperdiploid versus those with various fusion genes). If necessary the quality of this information can be further refined by enumerating chromosomes 14 with karyotyping or IGH alleles with interphase FISH. The data provide also insights into the biology of HeH leukemia suggesting that nondisjunction of chromosomes - leading to a HeH karyotype - affects a cell at the beginning of IGH recombination, which is a more undifferentiated B progenitor cell than the cell of origin of the TEL-AML1 positive leukemias and the group of other BCP ALLs.