E2A-ZNF384 and NOL1-E2A fusion created by a cryptic t(12;19)(p13.3; p13.3) in acute leukemia.

A 5-year-old boy who initially presented with ALL and relapsed 4 months later with AML was found to have an add(19) in the leukemia cells. FISH revealed that the add(19) was really a cryptic t(l2;l9)(p13.3;p13.3) interrupting E2A (TCF3). Nucleotide sequences of cloned genomic fragments with the E2A rearrangements revealed that the der(12) contained E2A joined to an intron of the NOLI (p120) gene. Reverse transcriptase (RT)-PCR of patient lymphoblast RNA showed expression of in-frame fusion cDNAs consisting of most of NOL1 fused to the 3' portion of E2A that encoded part of the second transcriptional activation domain and the DNA binding and protein dimerization motifs. The reciprocal der(19) E2A genomic rearrangements included 5' regions of E2A joined to an intron of the ZNF384 (NMP4, CIZ) gene, located approximately 450 kb centromeric to NOL1 on chromosome 12. RT-PCR showed expression of in-frame E2A-ZNF384 fusion cDNAs. To our knowledge, this is the second report of a chromosome translocation in leukemia resulting in two different gene fusions. This is the first report of expression of E2A fusion protein that includes the DNA binding and protein dimerization domains due to a more proximal break in E2A compared to those described previously.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase.

Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (Mer(Tg)) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the Mer(Tg) mice. At 12-24 months of age, greater than 55% of the Mer(Tg) mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in Mer(Tg) mice. Additionally, a significant survival advantage was noted in Mer(Tg) lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4--anaplastic lymphoma kinase gene fusion.

This report describes an unusual extramedullary hematologic malignancy in an 18-month-old child who presented with a capillary leak syndrome that evolved into hyperleukocytosis with malignant cells. The circulating tumor cells did not express an antigen profile typical of any subtype of leukemia commonly observed in children. Tumor cells were CD3(-)/CD56(+); had germline TCR genes; and strongly expressed CD30, epithelial membrane antigen, and anaplastic lymphoma kinase (ALK) consistent with a null cell anaplastic large cell lymphoma (ALCL). The malignant cells contained a t(2;19)(p23;p13.1) that interrupted ALK and translocated it to the der(19). Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis revealed fusion of ALK to tropomyosin 4, an ALK fusion partner not described previously in hematologic malignancies. The clinical presentation and phenotypic features of this malignancy were not typical for ALCL because tumor cells expressed both myeloid (CD13, CD33, HLA-DR) and natural killer (NK) cell antigens. The neoplastic cells most resembled NK cells because in addition to being CD3(-)/CD56(+) with germline TCR genes, these cells were CD25(+)/CD122(+)/granzyme B(+) and possessed the functional properties of immature NK cells. The unusual clinical presentation, immunophenotype, and functional properties of these neoplastic cells suggest that this malignancy may be derived from the putative myeloid-NK precursor cell. Furthermore co-expression of NK and ALCL features supports the concept that a minority of null-ALCL may be derived from NK cells and expands the spectrum of phenotypes that can be seen in tumors produced by ALK fusion proteins. (Blood. 2001;98:1209-1216)

Detection of E2A translocations in leukemias via fluorescence in situ hybridization.

Three rearrangements in ALL disrupt E2A and create E2A fusion proteins: the t(1;19)(q23;p13) and E2A-PBX1, t(17;19)(q22;p13) and E2A-HLF and a cryptic inv(19)(p13;q13) and E2A-FB1. While E2A is fused to PBX1 in most ALLs with a t(1;19), 5-10% of cases have translocations that appear identical, but do not affect E2A or PBX1. Because more intensive therapy improves the outcome of patients with E2A-PBX1positive (1;19) translocations, it is critical to identify this subset of patients so that appropriate therapy can be administered. In addition, there are balanced and unbalanced variants of the t(1;19) and controversy exists regarding the clinical significance of this distinction. We have developed a two-color fluorescence in situ hybridization assay that accurately detects E2A translocations in metaphase and interphase cells, distinguishes between balanced and unbalanced variants and identifies patients with a t(1;19) who lack E2A-PBX1 fusion. We found that clonal microheterogeneity is common in patients with E2A translocations and most patients have mixtures of cells with balanced and unbalanced translocations, suggesting that this distinction represents two ends of a continuum rather than distinct biological entities. These reagents should have widespread clinical utility and be useful for translational and basic research studies involving E2A translocations and this region of chromosome 19p13.

Protean clinical manifestations in children with leukemias containing MLL-AF10 fusion.

Translocations involving the MLL gene on chromosome 11q23 occur in 5-10% of human leukemias, and involve fusion with more than 30 different partner genes. The MLL-AF10 fusion produced by the t(10;11)(p12;q23) or ins(10;11)(p12;q23q13) occurs in a small percentage of acute leukemias, most commonly acute myelogenous leukemia (AML) of the M5 FAB subtype. We report two cases of AML (M5a and M0) and one case of acute lymphoblastic leukemia containing MLL-AF10 fusion. Each case had varied clinical characteristics, despite expressing similar MLL-AF10 fusion transcripts. Including the three cases described in this report, we identified a total of 38 cases of leukemia with MLL-AF10 fusion. Approximately one-third of these are not M5 AML. Taken together, these findings emphasize that while the sentinel molecular event may be identical in a disease, the clinical presentation and outcome can vary widely.

Oncogenesis in utero: fetal death due to acute myelogenous leukaemia with an MLL translocation.

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants <1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero. In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin-embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero.