Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes.

Cytogenetic studies have previously identified abnormalities of chromosome band 11q23 in many cases of infant acute leukemia. Recent studies by ourselves and others have demonstrated breakpoint clustering in acute leukemias bearing translocations involving 11q23, and a Drosophila trithorax gene homologue (called MLL, HRX, or ALL-1) has been shown to span the 11q23 breakpoints of these translocations. To determine if this gene is affected in infant acute myeloid leukemia (AML), we have analyzed 26 infant AML cases for molecular alterations of this 11q23 gene. 15 out of 26 cases studied (58%) showed rearrangement of the MLL gene at the molecular level, and these rearrangements were clustered within an approximately 11-kb region containing nine exons of this gene. Moreover, 14 of the 15 cases with 11q23 rearrangements (93%) had myelomonocytic or monocytic phenotypes (M4 or M5 FAB subtypes, respectively), both of which are associated with a poor prognosis in childhood AML. In contrast, only 1 of 11 nonrearranged cases had an M4 or M5 phenotype (P = 0.00002). Rearrangement also correlated significantly with hyperleukocytosis (P = 0.02), another clinical parameter associated with poor outcome in this disease. Our results demonstrate that molecular rearrangements of MLL are common in M4 or M5 infant AML, and suggest that alteration of this gene may result in abnormal control of proliferation and differentiation in monocytic progenitor cells.

Structural requirements of adenovirus VAI RNA for its translation enhancement function.

Recently, by genetic and biochemical approaches, it has been shown that adenovirus VAI RNA is required for efficient translation of viral mRNAs at late times after infection. To understand the nucleotide sequences and the domains of the VAI RNA that are responsible for the role of VAI RNA in enhancement of translation, a mutational analysis of the VAI gene was undertaken. Deletion, substitution, and insertion mutations covering most of the nucleotide sequences of VAI RNA were introduced into the VAI gene at the plasmid level. These mutant genes were then reintroduced into the virus, and growth properties of the mutant viruses were studied. The majority of the mutants retained normal or nearly normal levels of biological function. Mutations in the region between +43 and +53 and between +107 and the 3' end of the gene resulted in a considerable loss of activity. These mutants, however, grew significantly better than did an adenovirus type 5 mutant lacking both functional VAI and VAII genes, indicating that they retain a portion of their activity. Because no one mutation was able to completely abolish the function, we suggest that the VAI RNA may have multiple functional sites for its translation modulation function. These multiple sites may be short oligonucleotide sequences that may interact with cellular or viral components or both during translation.

Guanine-rich (GGNNGG) elements at chromosomal breakpoints interact with a loop-forming, single-stranded DNA-binding protein.

Proto-oncogene-activation is frequently preceded by chromosomal translocations. Several models suggest that DNA single-strands and loops may serve as intermediates in the process of illegitimate recombination. Guanine-rich, repetitive elements are preferred sites of chromosomal exchange and can undergo conformational changes which result in the generation of single-stranded DNA. Here we describe a single-stranded DNA-binding protein which binds specifically to guanine-rich elements at the breakpoints of human reciprocal translocations, including the t(14;18), t(2;8), t(9;22), t(15;17) and t(4;11) in leukemia and lymphoma. The primitive binding consensus consists of two guanine-residues on either side separated by a spacer of at least two nucleotides (GGN-NGG). Binding activity is unaltered by a spacer length of up to 46 nucleotides. These data suggest that the protein has the unique ability to form or stabilize DNA-loops and may thus play a general role in recombination.

Cloning and developmental expression of the murine homolog of the acute leukemia proto-oncogene AF4.

AF4 is the 4q21 gene involved in the acute lymphoblastic leukemia associated t(4;11)(q21;q23) where it forms a fusion gene with MLL. In order to gain insight into AF4's role in leukemogenesis we have studied its functional domains and expression pattern during murine development. We have cloned the murine homolog, Af4. We have demonstrated that 5' half of Af4 encodes a region with transcriptional transactivation activity which is disrupted by the t(4;11) in human leukemias. We have also localized the murine AF4 protein to the nucleus supporting a role for AF4 in transcription. The developmental expression pattern of Af4 was determined in situ hybridization and suggests Af4 plays an important role in the development of the hematopoietic, cardiovascular, skeletal and central nervous systems. A repeating pattern of Af4 expression in development is down-regulation with differentiation of a tissue. Among the cell types where this pattern of down-regulation is noted are B-lymphocytes. These findings raise the possibility that the disruption of normal AF4 function by the translocation may contribute to leukemogenesis.

MLL fusion partners AF4 and AF9 interact at subnuclear foci.

The MLL gene is involved in translocations associated with both acute lymphoblastic and acute myelogenous leukemia. These translocations fuse MLL with one of over 30 partner genes. Collectively, the MLL partner genes do not share a common structural motif or biochemical function. We have identified a protein interaction between the two most common MLL fusion partners AF4 and AF9. This interaction is restricted to discrete nuclear foci we have named 'AF4 bodies'. The AF4 body is non-nucleolar and is not coincident with any known nuclear structures we have examined. The AF4-AF9 interaction is maintained by the MLL-AF4 fusion protein, and expression of the MLL-AF4 fusion can alter the subnuclear localization of AF9. In view of other research indicating that other MLL fusion partners also interact with one another, these results suggest that MLL fusion partners may participate in a web of protein interactions with a common functional goal. The disruption of this web of interactions by fusion with MLL may be important to leukemogenesis.

Molecular analysis of 13 cases of MLL/11q23 secondary acute leukemia and identification of topoisomerase II consensus-binding sequences near the chromosomal breakpoint of a secondary leukemia with the t(4;11).

Rearrangements of the MLL (Mixed Lineage Leukemia) gene in the human 11q23 cytogenetic locus have been detected in secondary (therapy-related) acute leukemias in patients who have received topoisomerase II inhibitors for prior, independent neoplasms. The topoisomerase II inhibitors implicated in MLL/11q23 secondary leukemias all inhibit the religation step of reaction catalyzed by topoisomerase II. This results in the stabilization of a 'cleavable complex' with double-strand DNA breaks at the point of topoisomerase II binding. This raises the possibility that the cleavable complex participates in the translocation process in MLL/11q23 secondary leukemias. Here we report that the MLL/11q23 breakpoints in 13/13 patients with secondary leukemia map to the same breakpoint cluster region (bcr) noted in de novo MLL/11q23 acute leukemias and the presence of in vivo topoisomerase II inhibitor-induced cleavage sites in MLL/11q23 bcr. We have also cloned and sequenced the breakpoint from a MLL/11q23 secondary acute leukemia. This analysis revealed sequences similar to the consensus sequence for vertebrate topoisomerase II binding and cleavage close to the 11q23 and 4q21 breakpoints. These results support a role for topoisomerase II in mechanism generating translocations in MLL/11q23 secondary acute leukemia.

Pseudo-Gaucher histiocytes identified up to 1 year after transplantation for CML are BCR/ABL-positive.

The results of polymerase chain reaction (PCR) analysis after transplantation for chronic myelogenous leukemia (CML) are difficult to interpret clinically. Positive findings for BCR/ABL can be seen not only in patients who go on to relapse but also in patients who, after years of follow-up, remain in complete remission. The cause for the lack of concordance between PCR findings and relapse is not clear. We identified two patients with CML who had rare pseudo-Gaucher cells in their bone marrow aspirate specimens prior to, and at 1, and 6 or 12 months following syngeneic or allogeneic hematopoietic transplantation. After the transplant, the patients obtained clinical remission and were shown to be cytogenetically normal and to have germline MBCR in blood or bone marrow by Southern analysis. One patient was PCR-positive for BCR/ABL in the marrow at 12 months. In order to determine whether the pseudo-Gaucher histiocytes were BCR/ABL-positive, we used fluorescence in situ hybridization and probes for MBCR and ABL and analyzed Wright-stained smears to correlate molecular cytogenetic findings with cell type. On three aspirate smears from each patient (at 6 or 12 months post-transplant), all of the pseudo-Gaucher cells studied (10/10 in one patient and 12/12 in the other) showed the fusion for BCR/ABL. Other cells analyzed randomly (erythroid precursors, granulocytes and rare monocytes, lymphocytes and plasma cells) did not. Our cases provide the first proof that pseudo-Gaucher cells carry the BCR/ABL fusion. Furthermore, they illustrate that these cells can be found in the marrow for up to 12 months following transplantation. Our results permit speculation that pseudo-Gaucher cells or other long-lived histocytes may be one cause of persistent PCR positivity after transplantation that is not predictive of disease relapse.

Cloning and sequence analysis of four t(9;11) therapy-related leukemia breakpoints.

The t(9;11)(p22;q23) is the most common chromosomal translocation in topoisomerase II inhibitor therapy-related acute myeloid leukemia (tAML). This translocation fuses the MLL and AF9 proto-oncogenes producing a novel chimeric protein. In order to gain insight into the mechanism generating the t(9;11) and to clarify the role topoisomerase II inhibition may play in that mechanism we have cloned and sequenced the breakpoints from four tAML patients with the t(9;11). This sequence analysis identifies topoisomerase II consensus binding sequences near or at the chromosome 11 and chromosome 9 breakpoints in all four patients. One patient also had the consensus binding sequence for the TRANSLIN DNA-binding protein at the 9p22 and 11q23 breakpoints. Our results further support a direct role for topoisomerase II in the genesis of these tAML translocations.

Fatal thrombotic thrombocytopenic purpura despite plasmapheresis. A case report of autopsy findings.

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, fever and renal dysfunction. Prior to modern therapy, fewer than 3% of patients survived. However, with plasmapheresis, up to 82% of patients survive. We report a case of recurrent TTP which proved fatal despite plasmapheresis. At autopsy the hyaline thrombi found were notably smaller and of shorter duration than those of untreated TTP. These findings suggest that therapy had brought about an abatement of the microthrombotic process in spite of the fatal outcome.

Reactivity of human tissues with monoclonal antibodies to myeloid activation and differentiation antigens. An immunohistochemical study.

We have characterized antimyeloid monoclonal antibodies (mAbs) produced to human rheumatoid arthritis (RA) synovial tissue macrophages (MPs) (8D7) and to lipopolysaccharide (LPS)-treated U937 cells (3D8). The 3D8 antigen is upregulated with LPS stimulation of monocytes/MPs and during monocyte maturation. The 8D7 antigen is upregulated on functionally distinct subpopulations of RA synovial tissue MPs. We used immunohistochemistry to determine the spectrum of reactivity of these unique mAbs on myeloid cell suspensions, monocytes, and mature tissue inflammatory and noninflammatory MPs. The antigens identified by the mAbs were characterized biochemically, by immunoprecipitation of solubilized 125I-labelled antigens from cell surfaces, and immunohistochemically by enzymatic digestion of myeloid cells followed by a cellular ELISA. MAb 3D8, characterized as an anti-CD13 antibody, recognizes a 150-170 kd antigen, has almost exclusive myeloid reactivity, but reacts with Langerhans' cells of the skin and thymus, pointing to shared antigens between these cells and MPs. Unlike 3D8 antigen, 8D7 antigen is strongly expressed in inflammatory states, being present on MPs in granulomata as well as in sarcoid lymph nodes. Both mAbs react with frozen and methanol-Carnoy's fixed, paraffin-embedded tissues and detect antigenic differences among human mononuclear phagocytes present in different anatomical sites and in varying stages of differentiation and activation. These mAbs should prove to be a valuable tool for studying heterogenous populations of myeloid cells.

Monoclonal antibodies detect monocyte/macrophage activation and differentiation antigens and identify functionally distinct subpopulations of human rheumatoid synovial tissue macrophages.

Monoclonal antibodies (MAbs) to functionally heterogeneous populations of human rheumatoid arthritis (RA) synovial tissue macrophages and lipopolysaccharide (LPS)-activated U937 cells were generated. These MAbs were used to characterize macrophages in situ in the synovial pannus and to study relative antigen expression on the surface of cells isolated from the synovium and from normal peripheral blood. Monoclonal antibody 3D8, an anti-CD13 MAb, reacts with an antigen expressed on the surface of blood monocytes and is a monocyte activation-related antigen that is upregulated by exposure of monocytes to interferon-gamma (IFN-gamma) and LPS. The expression of the 3D8 antigen increases in parallel with MHC class II antigen expression and also is upregulated in culture as monocytes mature to macrophages. 3D8 antigen is expressed strongly on RA synovial tissue lining cells, which are thought to be composed of macrophages. 8D7 antigen expression, detected by MAb 8D7, increases on blood monocytes on cellular activation with LPS and interferon-gamma, but in contrast to the 3D8 antigen, does not increase with monocyte maturation in vitro. The 8D7 antigen is expressed differentially on density-defined macrophage subpopulations isolated from RA synovial tissue and is expressed more strongly on macrophages that are nonangiogenic than those that are angiogenic.

Yeast artificial chromosome cloning of a two-megabase-size contig within chromosomal band 18q21 establishes physical linkage between BCL2 and plasminogen activator inhibitor type-2.

The construction of large-scale physical maps requires efficient approaches to generate new probes and link informative markers. The mapping of a human chromosomal segment was initiated by using the 18q21.3 probes, plasminogen activator inhibitor type-2 (PLANH2) and BCL2, to screen a yeast artificial chromosome (YAC) library. An inverse polymerase chain reaction technique rescued genomic ends of the YAC inserts. These new probes were used in a chromosomal walking strategy which established that the PLANH2 gene was 600 kb telomeric and in the opposite transcriptional orientation to that of BCL2. Overall, 16 YACs with a mean size of approximately 300 kb were analyzed using rare-cutting restriction endonucleases and 10 end-rescued probes. A contiguous map within 18q21.3 that spans approximately 2 Mb was assembled. This establishes the feasibility of using YACs in the efficient cloning and physical surveying of expanses of the human genome lacking closely spaced, genetic landmarks.

Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene.

The common translocation found in human follicular lymphoma, t(14;18)(q32;q21), results in deregulation of the BCL2 protoonocogene. The isolation of the intact gene would provide an essential substrate to analyze the molecular basis of this malignancy. Pulsed-field gel electrophoresis suggested that this three-exon gene was several hundred kilobases (kb) long. Therefore, a library of yeast artificial chromosome (YAC) clones was screened to isolate the intact BCL2 gene. Two clones, yA85B6 (200 kb) and yB206A6 (700 kb), were isolated by using polymerase chain reaction (PCR) assays specific for exon I/II and exon III, respectively. However, neither YAC contained the entire BCL2 locus. Since the two YACs were found to overlap by 60 kb, we sought to take advantage of the high recombination frequency in yeast and induce physical recombination between the two clones. Cells containing each YAC were mated and induced to undergo meiotic division and sporulation. Analysis of the resulting tetrads revealed a spore containing a single recombinant YAC of 800 kb. PCR assays and Southern blotting demonstrated that this recombined YAC contained the entire approximately 230-kb BCL2 gene. Furthermore, probe order was conserved and there was no evidence of overt rearrangements or deletions. These results indicate the feasibility of reconstructing large genomic segments with overlapping YAC clones to study genes spanning hundreds of kilobases.

Monoclonal antibodies defining shared human macrophage-endothelial antigens.

We have selected several monoclonal antibodies (mAbs) producing using human rheumatoid arthritis (RA) synovial macrophages (m phi s) as immunogen. Of these, mAbs 8H2, 10G7 and 10G9 showed cross reactivity with endothelium, suggesting common antigens between these cell types. We have determined the spectrum of reactivity of these mAbs on hematopoietic cell lines, peripheral blood cells, and inflammatory and non-inflammatory tissues by immunohistochemistry. MAb 8H2 does not react with the myeloid cell lines HL60 (myelocytic), U937 (histiocytic lymphoma), and K562 (erythroleukemia), or with peripheral blood cells. In normal and inflamed tissue sections, mAb 8H2 reacts with m phi s and endothelial cells. In contrast, mAb 10G7 does not react with peripheral blood cells, but reacts with HL60, U937, and K562 cell lines, as well as with m phi s and endothelial cells in inflamed and noninflamed tissues. MAb 10G9 does not react with myeloid cell lines, but reacts with monocytes and platelets in peripheral blood. In both normal and inflamed tissues, mAb 10G9 reacts with m phi s and endothelial cells. The antigens identified by these three mAbs were characterized biochemically, by enzymatic digestion of RA synovial tissue m phi s followed by a cellular ELISA, as well as by reactivity of the mAbs with NIH-3T3 cells genetically engineered to express known myeloid antigens. These mAbs reacted with protein or glycoprotein antigens distinct from the known myeloid antigens CD13, CD14, CD33, CD34, CD36, and c-fms. These mAbs should prove to be a valuable tool for studying m phi s and endothelial cells and their shared antigenic determinants.

Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome.

Acute lymphoblastic leukemia (ALL) in infants generally shows distinctive biologic features and has a poor prognosis. Cytogenetic studies indicate that many infant leukemias have chromosome 11q23 translocations. Because of these findings and the distinct clinical features of infant leukemia, we investigated 30 cases of infant ALL for molecular defects of 11q23. Fourteen cases had cytogenetic abnormalities of 11q23, and all of them showed 11q23 rearrangements at the molecular level. An additional seven cases also had 11q23 molecular rearrangements, including one with normal cytogenetic analysis. Molecular abnormalities of 11q23 were significantly correlated with adverse prognostic factors, including age under 6 months, hyperleukocytosis, CD10- phenotype, and early treatment failure. Molecular analysis identified a group of infants with germline 11q23 that had a very good treatment outcome with a projected event-free survival of 80% at median follow-up of 46 months compared to 15% in infants with rearranged 11q23 (P < .001). These findings suggest that a high proportion (70%) of infants with ALL have 11q23 rearrangements and that these rearrangements are not always detectable by cytogenetic analysis. The presence of germline 11q23 DNA may identify a subgroup of infant ALL patients with a good outcome using current therapy and a different etiology for their ALL.

The chromosome 4q21 gene (AF-4/FEL) is widely expressed in normal tissues and shows breakpoint diversity in t(4;11)(q21;q23) acute leukemia.

The chromosomal translocation, t(4;11)(q21;q23), is the most common type of 11q23 chromosomal abnormality, being highly prevalent in infant acute leukemias and associated with a poor prognosis. The t(4;11) results in the fusion of an 11q23 gene (MLL, HRX, Htrx-1, or ALL-1) and a 4q21 gene (AF-4 or FEL). To further evaluate the 4q21 gene and its role in t(4;11) acute leukemia, we have cloned a 38-kb genomic region and mapped exons of the AF-4 gene. The 4q21 breakpoints in 19 cases of t(4;11) acute leukemia were analyzed by Southern analysis and pulsed-field gels. Seventeen of the 19 cases had breakpoints on chromosome 4q21 that were scattered in this 38 kb region. Expression of the AF-4 gene was studied in a total of 28 various nonhematopoietic, hematopoietic, and t(4;11) leukemic cell lines. The AF-4 gene was expressed in all cell lines as a major and a minor transcript. In addition to the normal transcripts, two fusion transcripts from the derivative 11 and derivative 4 chromosomes were identified in all t(4;11) cell lines except B1, which had only the der(11) transcript. These findings suggest that the breakpoints on 4q21 cluster over a broader area than do the breakpoints in the 11q23 gene, and that der(11) encodes the fusion RNA found consistently in leukemia cells.

Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product.

A chromosomal translocation, t(4;11)-(q21;q23), is associated with an aggressive mixed-lineage leukemia. A yeast artificial chromosome was used to clone the chromosomal breakpoint of this translocation in the RS4;11 cell line. The breakpoint sequences revealed an inverted repeat bordered by a consensus site for topoisomerase II binding and cleavage as well as chi-like elements. The der(11) chromosome encodes a fusion RNA and predicted chimeric protein between the 11q23 gene MLL and a 4q21 gene designated AF4. The sequence of the complete open reading frame for this fusion transcript reveals the MLL protein to have homology with DNA methyltransferase, the Drosophila trithorax gene product, and the "AT-hook" motif of high-mobility-group proteins. An alternative splice that deletes the AT-hook region of MLL was identified. AF4 is a serine- and proline-rich putative transcription factor with a glutamine-rich carboxyl terminus. The composition of the complete MLL-AF4 fusion product argues that it may act through either a gain-of-function or a dominant negative mechanism in leukemogenesis.

The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia.

The t(4;11)(q21;q23) is the most common translocation involving band 11q23 and is found predominantly in acute lymphoblastic leukemias (ALLs) of infants. Recent studies have shown that this translocation involves the MLL gene on chromosome 11 and the AF-4 gene on chromosome 4. Using oligonucleotide primers derived from these genes, we established reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of the fusion transcripts from both the der(11) and der(4) chromosomes. Using these assays we analyzed 23 pediatric cases of t(4;11) containing ALL. RT-PCR analysis for the der(11)-derived MLL/AF-4 fusion transcript resulted in its detection in every case at a sensitivity of greater than 1 leukemic cell in 10(5) cells. Sequence analysis of MLL/AF-4 PCR products demonstrated fusion mRNAs resulting from breaks in MLL introns 6, 7, or 8, with alternative splicing to one of three exons in the AF-4 gene. In contrast, analysis for the der(4)-derived transcript resulted in the detection of this chimeric mRNA in only 84% of the cases analyzed. These data suggest that the critical chimeric gene product involved in the establishment of the leukemic clone is derived from the der(11) chromosome. Moreover, these data demonstrate the utility of the RT-PCR assay for the der(11)-encoded message both for diagnosing t(4;11)-containing leukemia and for monitoring patients for minimal residual disease.