Clonal characteristics of acute lymphoblastic cells derived from BCR/ABL p190 transgenic mice.

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (JH) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL).

Absence of p53 permits propagation of mutant cells following genotoxic damage.

Much evidence has been gathered in support of a critical role for p53 in the cellular response to DNA damage. p53 dysfunction is associated with progression and poor prognosis of many human cancers and with a high incidence of tumours in p53 knockout mice. The absence of a p53-dependent G1 arrest that facilitates DNA repair or apoptosis might impact critically on clinical cancer in two ways. First, by abrogating the impact on therapy that operates via genotoxic damage and apoptosis; and second, by encouraging progression either by inducing genomic instability and DNA mis-repair or by permitting survival of mutants. However, experiments examining the relationship between p53 deficiency and mutation frequency have so far failed to confirm these predictions. The precise role played by p53 is therefore unclear. We now report use of a short term in vitro approach to assess the influence of p53 on radiation-induced mutations at the hprt locus in murine B cell precursors that are normally radiation ultrasensitive. We find a high number of hprt mutants among X-irradiated p53 null cells, which results from preferential survival as clonogenic mutants rather than from a p53-dependent increase in mutation rate. This result has important implications for genotoxic cancer therapy.

A myeloid-lineage-specific enhancer upstream of the mouse myeloperoxidase (MPO) gene.

The myeloperoxidase (MPO) gene is selectively expressed during haemopoiesis in the granulocytic lineage. Compared with the erythroid (beta-globin) and B-cell (immunoglobulin) lineages, little is known of the regulatory sequences and transcription factors involved in the regulation of genes specific for granulopoiesis. We have approached this issue by identifying a strong enhancer for the murine MPO gene. A candidate enhancer region was mapped by the detection of a strong DNase I hypersensitive site, -3.4 to -3.2 kb upstream of the MPO gene. A 301 bp fragment encompassing the DNase I site was shown to have strong enhancer function in a transient assay following transfection of a reporter gene into a MPO-expressing cell (WEHI 3BD+), but was inactive in lymphoid cells. Analysis of sub-fragments revealed that the whole 301 bp fragment is required for maximal enhancer function.

Simultaneous adult T-cell leukemia/lymphoma and sub-acute polyneuropathy in a patient from Chile.

This paper reports a case of adult T-cell leukemia/lymphoma associated with human T-cell lymphotropic virus type I (HTLV-I) diagnosed in a Chilean patient who developed after 1 1/2 years a crisis with a progressive sensorimotor polyneuropathy. Serum and cerebrospinal fluid HTLV-I antibody tests were positive and HTLV-I DNA was clonally integrated in peripheral lymphocytes. This case is unusual in having simultaneous neurological disease. Along with other recent data from South America, this suggests that the endemic area of HTLV-I may spread far beyond the Caribbean area.

Lineage specificity of rearrangement and expression of genes encoding the T cell receptor-T3 complex and immunoglobulin heavy chain in leukemia.

In acute lymphoblastic leukemia (ALL) diagnostic samples and cell lines with unequivocal B cell precursor (common) or T cell precursor immunophenotypes, there is inappropriate or cross-lineage IgH or T cell receptor beta gene (TCR beta) rearrangement in approximately 25% of the cases. The frequency of such rearrangements is lower in mature lymphoid neoplasms and acute myeloblastic leukemia. The most immature B lineage ALL ('null' ALL) has a much lower frequency of TCR gene rearrangement than the common variant of B cell precursor ALL and also has a high frequency of oligoclonal rearrangements of IgH genes. Non-T leukemic cells with inappropriately rearranged TCR beta gene did not necessarily have a rearranged TCR gamma gene. Inappropriately rearranged IgH or TCR genes are usually not expressed at the mRNA level, and the gene for the TCR associated protein T3 delta is not detectably expressed at the mRNA or protein level in leukemias classified unambiguously as non-T. Five cases of acute leukemia with ambiguous or mixed lineage immunophenotypes (myeloid + T or myeloid + B) are described. These five had diverse patterns of IgH, TCR beta, and TCR gamma rearrangement, and all expressed terminal transferase concomitantly with MY9 (CD33). The T3 delta gene was expressed in two cases, which also expressed other T cell markers indicating that coordinated lymphoid lineage programs had been initiated. The implications of these observations for lineage-associated regulation of genes during normal differentiation and leukemogenesis are discussed.

Rapid intraclonal switch of lineage dominance in congenital leukaemia with a MLL gene rearrangement.

We describe a case of neonatal mixed lineage leukaemia which presented with a dominant B progenitor lymphoblast population plus a minor monocytic component. Treatment of the patient with corticosteroid and Ara-C resulted in loss of lymphoblasts and a rapid (within 7 days) increase and dominance of the monocytic component. The common clonal origin of the two cell types was evident from the identical rearrangement in the MLL gene and a shared rearrangement of one IGH allele. In common with other neonatal or infant ALL with MLL gene rearrangements, this leukaemia may have originated in a common B-monocytic lineage stem cell during foetal haemopoiesis. The observations further suggest that the therapeutic impact of the MLL gene rearrangement is to some extent dependent on the cellular context in which it is expressed.

HTLV-I positive adult T-cell leukaemia/lymphoma (ATLL) in Chile.

We describe the clinical and laboratory features of nine patients born in Chile with HTLV-I-positive adult T-cell leukemia/lymphoma (ATLL). All were adults (median age 51 years) of Caucasian origin without evidence of Indian or foreign extraction and none had been out of the country. The main disease features were organomegaly, cutaneous lesions, hypercalcemia and leukemia with atypical polylobed lymphocytes displaying a CD2+, CD3+, CD4+, CD8-, CD7- T-cell phenotype. Eight patients presented with acute type ATLL and one had a chronic form lasting for 16 months prior to the development of the acute phase. Lymph node histology (three cases) was consistent with a T-cell non-Hodgkin's lymphoma (large and small cells). Antibodies to HTLV-I were detected by ELISA and particle agglutination in the serum from eight of nine patients. DNA analysis showed HTLV-I proviral DNA in all seven cases investigated, including the single serologically negative patient. In five cases, HTLV-I was monoclonally integrated and in one case oligoclonal. In the seventh case viral DNA clonal status was ambiguous. Response to therapy was poor and median survival was 3 months (range 2-20 months). This study provides further evidence that HTLV-I is endemic in Chile, a non-tropical country where the two main diseases associated with HTLV-I, ATLL and TSP, are found.

Screening for herpesvirus genomes in common acute lymphoblastic leukemia.

There is epidemiological evidence that infection may play a role in the etiology of childhood leukemia in particular common B cell precursor acute lymphoblastic leukemia. A panel of 20 leukemic samples (panel 1) was examined for the presence of four lymphotropic herpesviruses using conventional molecular techniques. A second independent panel of 27 leukemic samples (panel 2), along with 28 control peripheral blood samples from children with other forms of cancer, was tested for the presence of the same four viruses using sensitive real-time quantitative PCR. While herpesvirus genomes were detected, they were present at very low levels; detection rates and levels were similar in the leukemic and control panels. In addition we surveyed 18 leukemic samples (five from panel 1, six from panel 2 and a further seven samples not previously analyzed) using a degenerate PCR assay capable of detecting the genomes of known herpesviruses plus putative new members of the family. No novel herpesvirus genomes were detected suggesting that a herpesvirus is unlikely to be etiologically involved as a transforming agent in common acute lymphoblastic leukemia.

Protracted dormancy of pre-leukemic stem cells.

Cancer stem cells can escape therapeutic killing by adopting a quiescent or dormant state. The reversibility of this condition provides the potential for later recurrence or relapse, potentially many years later. We describe the genomics of a rare case of childhood BCR-ABL1-positive, B-cell precursor acute lymphoblastic leukemia that relapsed, with an acute myeloblastic leukemia immunophenotype, 22 years after the initial diagnosis, sustained remission and presumed cure. The primary and relapsed leukemias shared the identical BCR-ABL1 fusion genomic sequence and two identical immunoglobulin gene rearrangements, indicating that the relapse was a derivative of the founding clone. All other mutational changes (single-nucleotide variant and copy number alterations) were distinct in diagnostic or relapse samples. These data provide unambiguous evidence that leukemia-propagating cells, most probably pre-leukemic stem cells, can remain covert and silent but potentially reactivatable for more than two decades.

Evolutionary trajectories of hyperdiploid ALL in monozygotic twins.

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally.

Clonal origins of ETV6-RUNX1⁺ acute lymphoblastic leukemia: studies in monozygotic twins.

Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage.

Exon scrambling of MLL transcripts occur commonly and mimic partial genomic duplication of the gene.

The MLL gene is frequently rearranged in acute human leukemia of both the myeloid and lymphoid lineages. Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we identified several abnormally spliced transcripts in which MLL exons were joined in an order different from the genomic orientation (scrambled exons). Mis-splicing of MLL was present in both normal and malignant tissues. Although the majority of these scrambled transcripts were joined accurately at consensus splice sites, there were several examples in which the junctions of exons spliced in aberrant order were at non-consensus sites. A number of features differentiate mis-splicing of MLL from the previously described cases of scrambled exons and circular RNAs. Some scrambled transcripts appear to be present in the polyadenylated fraction of RNA. No correlation of exon scrambling with exon skipping was found, and there was no particular tendency for the exons involved to be near large introns. Our data show that splicing of MLL is extremely complex. The presence of scrambled transcripts in both normal and leukemic cells, indistinguishable from transcripts resulting from genomic MLL rearrangements, precludes the use of nested RT-PCR as a screening method for detection of tandem duplication of tandem duplication of MLL.

Multiple changes in chromatin structure precede the transcriptional activation of the human growth hormone locus in placental cells.

In addition to the growth hormone gene (hGH-N) itself, the human growth hormone (hGH) locus contains four related genes, namely hGH-V and hCS-L, -A and -B, which have appeared very recently in evolution and are specifically expressed in placenta. With the aim of identifying the regulatory elements responsible for this placental-specific expression, we have mapped the DNaseI hypersensitive sites present at the hGH gene cluster in a placental cell line (BeWo) that expresses the hGH-V and hCS genes. Our results reveal a complex pattern of hypersensitive sites distributed along the hGH locus, most of which appear to be cell type-specific. Thus, we have identified placental-specific hypersensitive sites within the first intron of the hGH-N and hGH-V genes, but not in the equivalent regions of the hCS genes. In addition, we have found several placental-specific hypersensitive sites downstream of the hCS-L and hCS-A genes, which might reflect the presence of enhancer elements similar to that located downstream of the hCS-B gene (Walker et al. (1990) J. Biol. Chem. 265, 12940). Comparison of BeWo cells with a placental cell line (JEG-3) which does not express the hGH-V and hCS genes revealed a very similar pattern of hypersensitive sites, suggesting that the sites detected are established before the onset of transcription. Our results indicate that the transition to an active hGH locus in placental cells requires multiple alterations in chromatin structure, and provide a framework for the molecular analysis of the regulatory elements and mechanisms mediating such processes.

Protein expression and constitutive phosphorylation of hematopoietic transcription factors PU.1 and C/EBP beta in acute myeloid leukemia blasts.

The transcriptional activity of transcription factors is regulated by phosphorylation. The uncontrolled expression and constitutive activation of transcriptional regulators have been reported to cause malignant diseases. However, little is known about the phosphorylation status of tissue-specific transcription factors in human primary malignancies. Here we present the first insights into both protein expression and phosphorylation of transcription factors in a large-scale study of patients with acute myeloid leukemia (AML). We examined the expression and phosphorylation status of hematopoietic transcription factors PU.1 and C/EBP beta detected by the retarded mobility of the phosphorylated forms of the proteins. The rate of protein expression differed among French-American-British (FAB) subclasses. The expression of C/EBP beta and PU.1 were detectable in 77% and 61%, respectively, of 90 AML samples examined. The expressed PU.1 and C/EBP beta was always accompanied with both phosphorylated and unphosphorylated forms of PU.1 and C/EBP beta, respectively. Statistical significance was observed between PU.1 expression (phosphorylation) and FAB classification (M0, M4, or M5 versus M2 or M3, P < .0001). PU.1 and C/EBP beta were simultaneously detected in all M0, M4, M5 and peripheral blood monocytes, whereas in M2 and M3, the expression of the 2 transcription factors varied among samples. Examination of protein expression and phosphorylation of these lineage-specific molecules may help us to understand the functional characteristics of AML.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

HTLV-I positive progressive spastic paraparesis (TSP) associated with a lymphoid disorder in three Chilean patients.

We describe the clinical and laboratory features in three Caucasian Chilean patients with tropical spastic paraparesis (TSP) associated with/or preceded by a lymphoproliferative disorder involving cutaneous lesions and localised lymphadenopathy. The neurological symptoms and signs were characteristic of TSP and CSF examination revealed the presence of oligoclonal bands. All three patients had a moderate leucocytosis (10-14 x 10(9)/l) with eosinophilia and a minority (2-4%) of circulating atypical polylobed or ATLL-like lymphocytes. Lymph node histology showed a diffuse pattern of infiltration (1 case) and marked expansion of the paracortical zone with convoluted lymphocytes and immunoblasts (2 cases). Skin biopsy demonstrated a dermal lymphoid infiltration with epidermotropism. Antibodies to HTLV-I were detected in the serum and CSF in the three patients and Southern blot analysis of peripheral blood mononuclear cells showed a monoclonal integration of HTLV-I proviral DNA in one case whereas in the two others the pattern was indicative of low level polyclonal integration. All three patients were treated with prednisolone and one with PUVA with transient partial response on the skin and neurological manifestations. Two patients died months to 5 years from presentation and the other is alive 12 years from diagnosis with active neurological and skin disease. The simultaneous occurrence of HTLV-I associated TSP with smouldering ATLL and a cutaneous ATLL or pre-leukaemic form is discussed.

Influence of salinomycin on antimicrobial resistance of coliforms and streptococci from broiler chickens.

An experiment was conducted to evaluate antimicrobial resistance of coliforms and streptococci isolated from feces of chickens fed salinomycin. Two groups of 20 chickens were fed either a control feed or feed supplemented with 80 g/ton salinomycin. Chicken fecal coliforms and streptococci were isolated at 5, 15, 19, 22, 26, 33, 40, and 47 days of age and their resistance to 11 or 12 antibacterial agents (coliforms and streptococci, respectively) were determined in both groups of chickens. Salinomycin significantly reduced the number of coliforms resistant to sulfadiazine and reduced the number of streptococci resistant to erythromycin and lincomycin. Streptococci from birds fed salinomycin had lower minimum inhibitory concentrations for streptomycin. No streptococci isolates developed resistance to salinomycin. Coliforms from birds fed salinomycin had more (P less than .05) resistance patterns involving two, five, and six drugs. Numbers of coliforms resistant to streptomycin, ampicillin, carbenicillin, and cephalothin were greater (P less than .05) from birds fed salinomycin.

Influence of salinomycin on incidence, shedding, and antimicrobial resistance of Salmonella typhimurium in experimentally infected broiler chicks.

Twenty broiler chickens were fed 80 g/T salinomycin, an antibiotic produced by Streptomyces albus, and 20 birds were fed a control, unmedicated feed. The birds were experimentally infected with Salmonella typhimurium. The study evaluated the effects of salinomycin on Salmonella incidence, shedding, and antimicrobial resistance. Salinomycin had no effect on body weights, length of time salmonellae were shed, number of salmonellae shed on postdosing day 3, salmonellae tissue recoverability, or on the total number of resistance patterns. Salinomycin caused the decline of salmonellae to be more gradual; however, both treatments were comparable at the end of the study. The majority of isolated from birds receiving salinomycin maintained the original S. typhimurium antibiogram of streptomycin, sulfadiazine, and nalidixic acid. The salinomycin salmonellae were more susceptible to tetracycline, amikacin, carbenicillin, gentamicin, and cephalothin. The multiple resistance patterns of eight and nine drugs tended to be more prevalent among salmonellae from control birds than salinomycin treated birds. The antibiotic salinomycin appears to be an acceptable feed additive in broilers at the level of 80 g/T based on these results of its effects on salmonellae shedding and antimicrobial resistance.

Activated sludge flocculation: direct determination of the effect of calcium ions.

The effect of calcium on activated sludge flocculation dynamics is investigated using a unique experimental technique. The technique allows on-line analysis of the size of activated sludge flocs during flocculation and provides valuable insight into the mechanisms of flocculation. Activated sludge samples were firstly sonicated for 3 minutes at 50 W and then stirred at 100 rpm. The floc size was subsequently measured on-line using a Malvern Mastersizer/E. For concentrations of calcium less than 4 meq/L no significant increase in final floc size was observed even though an increase in the initial rate of change of floc size could be seen. Addition of calcium greater than 4 meq/L resulted in a dramatic increase in floc size. Results from this investigation support the theory that cations are involved in flocculation through cationic bridging, and will be used in ongoing investigations to model the flocculation process.

Practice pattern variation among internal medicine specialists in the prevention of glucocorticoid-induced osteoporosis.

Osteoporosis is a serious side effect of long-term glucocorticoid (GC) use, but there is little success at prevention. We sought to identify academic physicians' awareness of glucocorticoid-induced osteoporosis (GIOP) risk and the patient and provider characteristics that determine GIOP management. A retrospective chart review of 365 patients seen at The University of Alabama at Birmingham by 4 rheumatologists, 3 pulmonologists, and 3 gastroenterologists was performed. Of these, 59.2% were women and 69.3% were Caucasians. Only 110 patients (30.1%) received any type of GIOP prevention intervention. The patients receiving GIOP prevention were older (58.7 +/- 13.8 vs. 49.8 +/- 16.7 years; p < 0.001); had longer duration of GC use (91.9 +/- 84.9 vs. 50.0 +/- 57.7 months; p < 0.001); and, for women, were more likely post-menopausal (81.5% vs. 18.5% premenopausal; p < 0.001). Fracture history was more common in those who received GIOP management (18 vs. 9 cases; p < 0.001). Calcium was the most commonly prescribed prevention strategy (84.5%). Recommendation of risk factor modification was seldom documented. Using multivariate logistic regression, rheumatologists were 4 times more likely to recommend GIOP prevention than the other two specialists. To improve the education in GIOP prevention strategies for specialists who commonly prescribe long-term GC, regular meetings and guidelines provided by experts in this field should be conducted. Both risk factors modification and pharmacological intervention for GIOP prevention should be started at the time of first GC prescription or as early as possible.