Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients.

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.

Single site polymorphisms and alternative splicing of the human CD13 gene--different splicing frequencies among patients with acute myeloid leukaemia and healthy individuals.

Within the haematopoietic system, CD13/aminopeptidase N (APN), a transmembrane glycoprotein, is expressed on the surface of early committed progenitors of granulocytes and monocytes and by all cells of these lineages as they mature. CD13 is expressed on the majority of leukaemic myeloblasts in acute myeloid leukaemia (AML), and on leukaemic lymphoblasts in a small percentage of acute lymphoid leukaemia cases. Thus, anti-CD13 monoclonal antibodies are used as diagnostic markers in leukaemia typing. By systematically amplifying overlapping reverse transcription polymerase chain reaction (RT-PCR) amplicons throughout the CD13 mRNA, we identified two splice variants in which exon 3 and exon 14 were lost. Fourteen healthy individuals and 34 patients with AML were screened for these splice variants. All healthy individuals, and the majority of AML patients, had both splice variants but they represented less than 10% of the total RT-PCR-amplified CD13 product. Increased expression of both truncated CD13 mRNA forms were observed in 6% of AML patients, whereas no detectable exon 3 or exon 14 splice variants could be generated in 26% and 9% of AML patients respectively. The different splicing frequencies may reflect altered processing of pre-mRNA or expansion of certain cell types for some AML patients, even though no correlation existed to blast percentage, FAB classification, surface antigens or cytogenetic characteristics. In addition, we identified an intron of 506 bp between exon 1 and exon 2 as well as two sites of single nucleotide polymorphism with a heterozygosity index of about 0.5, making them useful as genetic markers.

FAB M4 and high CD14 surface expression is associated with high cellular resistance to Ara-C and daunorubicin: implications for clinical outcome in acute myeloid leukaemia.

In 145 adult patients diagnosed with non-M3 acute myeloid leukaemia (AML) the relevance of FAB-subtype and immunophenotype to in vitro cellular drug resistance towards the anthracyclines aclarubicin (Acla) and daunorubicin (Dau), and the nucleoside analogue cytarabine (Ara-C), as well as other antileukaemic drugs, was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We demonstrate that high CD14 expression is highly significantly associated with high cellular Ara-C and Dau resistance in univariate as well as multivariate analyses. FAB subtypes with highest and lowest cellular Ara-C resistance were M4 and M5, respectively (P < 0.01, one-way anova), whereas FAB subtypes with highest and lowest cellular Dau resistance were M4 and M1, respectively (P < 0.01, one-way anova). By contrast, no significant differences in cellular drug resistance towards Acla could be demonstrated among FAB subtypes. Furthermore, in two cohorts of AML patients treated by two different regimens for remission induction over a period of 15 yr (1985-94, n = 159 and 1995-99, n = 76, respectively) we demonstrate in univariate analyses a significance of CD14 expression with respect to clinical outcome. With the exception of significance to probability of obtaining complete remission in the first cohort (P = 0.03, logistic regression), this significance was, however, lost in multivariate analyses. It was demonstrated that FAB-M4 patients were older than M5 patients and that high CD14 expression was associated with the presence of secondary AML and older age. We conclude that although cases with high blast cell CD14 expression (and FAB-M4 cases) were more resistant to Ara-C as well as Dau in vitro, the clinical and biological significance of this may be debatable because of interactions with major prognostic factors in AML.

Leukaemia cell drug resistance and prognostic factors in AML.

In 93 cases of acute myeloid leukaemia (AML) the extent to which prognostic factors mirrored the in vitro cellular chemotherapy resistance (to anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as nucleoside analogue cytarabine (Ara-C)) was investigated using a 4-d MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. We found that age at presentation and presence of secondary AML were significantly correlated to leukaemia cell Ara-C resistance. Thus, analysis of in vitro drug resistance data revealed that age at presentation and presence of secondary leukaemia were both independently correlated to cellular drug resistance, with older age being associated with higher Ara-C resistance in vitro (p=0.02 and 0.01 in univariate and multivariate analyses, respectively) and with secondary leukaemia being associated with higher Ara-C resistance (p=0.04 and 0.059 in univariate and multivariate analysis, respectively). Median LC-50 values (Ara-C) were: 178 ng/ml in paediatric cases, 356 ng/ml in younger adult cases, and 584 ng/ml in elderly (age > or = 60 yr) cases giving a resistance ratio between these age subgroups of 1:2.0:3.3. Median LC-50 values (Ara-C) was 381 ng/ml in de novo cases as opposed to 891 ng/ml (resistance ratio 1:2.3) in secondary cases. By contrast, cytogenetic findings, presenting leucocyte count, FAB-subtype, and gender were not consistently correlated to in vitro drug resistance to any of the three drugs. We conclude that at least two major adverse prognostic factors in AML (advanced age at presentation and presence of secondary leukaemia) are associated with increased leukaemia cell Ara-C resistance. High leucocyte count is not associated with increased cellular drug resistance towards Acla, Ara-C or Dau.

[Acute myeloid leukemia. Therapeutic results 1985-1994]. / Akut myeloid leukaemi. Behandlingsresultater 1985-1994.

In a retrospective study we evaluate the treatment and outcome of 421 adults admitted to our department with acute myeloid leukemia (AML) during the 10 year period from 1985-1994. Younger patients (< or = 55 years) had a significantly better prognosis than elderly patients (> 55 years), partly because more younger patients had remission-induction therapy (81% versus 39%) and their complete remission (CR) rate was higher (69% versus 41%). In patients achieving CR the long-term survival (five years) was 40% for younger and 26% for elderly patients. Nineteen patients received autologous bone marrow transplantation (BMT) and eight an allogeneic BMT in first CR. Five patients treated with allogeneic BMT are still relapse-free, whereas autologous BMT, in comparison to conventional chemotherapy alone, did not improve the five-year relapse-free survival (29 versus 27%). Our data illustrate that in a non-selected material of AML patients the long-term survival (five years) has only slightly improved. The effect of autologous BMT cannot be evaluated in this retrospective setting.

Studies on the urea cycle enzyme ornithine transcarbamylase using heavy atom isotope effects.

Ornithine transcarbamylase (OTCase) catalyzes the reaction between L-ornithine and carbamyl phosphate in the first step of the urea cycle. 13C isotope effects were measured in carbamyl phosphate, using OTCase obtained from E. coli in a one-column purification which yielded 30 mg of very pure enzyme from 51 of cell culture. At near zero L-ornithine, the 13C kinetic isotope effect was 1.0095, at high levels of L-ornithine (86 mM) the 13C kinetic isotope effect was unity, and 0.83 mM ornithine was found to eliminate half the isotope effect. These results are indicative of an ordered kinetic mechanism in which carbamyl phosphate binds to the enzyme before L-ornithine. Similar experiments were performed using the slow substrate L-lysine in place of L-ornithine. At 90 mM L-lysine the 13C kinetic isotope effect was large, 1.076. This value is most likely the intrinsic kinetic isotope effect with this substrate, and the chemistry of the enzyme catalyzed reaction has become rate limiting.

[10 years' experiences with flow cytometric immunological phenotyping in malignant hematologic diseases]. / Ti års erfaring med flowcytometrisk immunologisk faenotypebestemmelse ved maligne haematologiske sygdomme.

Immunological phenotyping of mononuclear cell suspensions from bone marrow and/or peripheral blood from 2341 patients (6140 examinations) suspected of malignant haematological diseases was performed during a 10-year period. The cells were labelled with a panel of monoclonal antibodies and subjected to flowcytometry. The results showed a clear distinction between acute lymphoblastic leukaemia and acute myeloid leukaemia, and a new group of acute hybrid leukaemia (3.3% of all acute leukaemia cases) was established. During the 10-year period immunological phenotyping has changed from being a research method to a widely employed method for cell characterisation early in the course of malignant haematological diseases.

Role of glucagon in maintenance of euglycemia in fed and fasted rats.

The role of glucagon in the regulation of blood glucose in fed and fasted anesthetized rats was studied by injecting intravenously 4 ml/kg of a high-capacity (40 nmol/ml) high-affinity (0.6 x 10(11) mol/l) monoclonal glucagon antibody. Blood glucose was lowered by the antibody by 2 mmol/l in fed rats but remained unchanged in 10- and 48-h-fasted rats. Antibody injection significantly reduced plasma insulin in both fed and 10-h-fasted rats. In 10-h-fasted rats, propranolol injection decreased blood glucose by 0.6 mmol/l, and combined with antibody administration, a decrease by 1.1 mmol/l was observed. Blood glucose was never < 3.3 mmol/l. Thus glucagon is partly responsible for maintenance of euglycemia in fed rats, whereas during fasting it plays a limited role. However, immunoneutralization of glucagon reduces insulin secretion irrespective of blood glucose. Additional mechanisms seem to be responsible for the maintenance of blood glucose in the fasting state when glucagon and the sympathoadrenergic system are blocked.

Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats.

The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ml), high-affinity (0.6 x 10(11) l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p < 0.05, n = 6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2 +/- 0.7 mmol/l to 17.3 +/- 1.8 mmol/l in controls vs 10.5 +/- 0.9 mmol/l to 9.3 +/- 1.0 mmol/l; cross-over: n = 6, p < 0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n = 4) and in severely hyperglycaemic rats injected with STZ as adults (n = 6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7 +/- 4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0 +/- 3.8 mmol/l; cross-over: n = 5, p < 0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.

Effects of non-esterified fatty acids on insulin-stimulated glucose transport in isolated skeletal muscle from patients with type 2 (non-insulin-dependent) diabetes mellitus.

The influence of elevated levels of oleate on insulin-stimulated 3-0-methylglucose transport was assessed in vitro, in isolated skeletal muscle obtained from patients with type 2 (non-insulin-dependent) diabetes mellitus (n = 7) and control subjects (n = 8). An increase in oleate levels from 0.3 to 1.0 mmol/l induced a 3.7-fold increase in the rate of oleate oxidation (P < 0.01) in skeletal muscle from control subjects. However, the rate of insulin-stimulated 3-0-methylglucose transport was not altered in isolated skeletal muscle from the control subjects or the type 2 diabetic patients following exposure to 1.0 mmol/l oleate. This observation indicates that elevation of non-esterified fatty acids to a high physiological level has no inhibitory effect on glucose transport.

Effects of glycaemia on glucose transport in isolated skeletal muscle from patients with NIDDM: in vitro reversal of muscular insulin resistance.

We investigated the influence of altered glucose levels on insulin-stimulated 3-0-methylglucose transport in isolated skeletal muscle obtained from NIDDM patients (n = 13) and non-diabetic subjects (n = 23). Whole body insulin sensitivity was 71% lower in the NIDDM patients compared to the non-diabetic subjects (p < 0.05), whereas, insulin-mediated peripheral glucose utilization in the NIDDM patients under hyperglycaemic conditions was comparable to that of the non-diabetic subjects at euglycaemia. Following a 30-min in vitro exposure to 4 mmol/l glucose, insulin-stimulated 3-0-methylglucose transport (600 pmol/l insulin) was 40% lower in isolated skeletal muscle strips from the NIDDM patients when compared to muscle strips from the non-diabetic subjects. The impaired capacity for insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle was normalized following prolonged (2h) exposure to 4 mmol/l, but not to 8 mmol/l glucose. Insulin-stimulated 3-0-methylglucose transport in the NIDDM skeletal muscle exposed to 8 mmol/l glucose was similar to that of the non-diabetic muscle exposed to 5 mmol/l glucose, but was decreased by 43% (p < 0.01) when compared to non-diabetic muscle exposed to 8 mmol/l glucose. Despite the impaired insulin-stimulated 3-0-methylglucose transport capacity demonstrated by skeletal muscle from the NIDDM patients, skeletal muscle glycogen content was similar to that of the non-diabetic subjects. Kinetic studies revel a Km for 3-0-methylglucose transport of 9.7 and 8.8 mmol/l glucose for basal and insulin-stimulated conditions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of a method for simultaneous analysis of cell surface phenotype and cell cycle in human bone marrow and peripheral blood leukocytes by flow cytometry: the value of both internal and external standards.

Three different methods for the simultaneous analysis of surface phenotype and DNA quantification were compared. One method, involving the fixation of cells in 70% ethanol, was convincingly superior, both with regard to the CV of the G0G1 peak and the intensity of the DNA labelling. Furthermore, the correlation between the surface antigen densities before and after fixation were high. Experiments evaluating the intraday and the interday variation of the DNA ratio (the mean channel of the G0G1 peak of the sample divided by the mean channel of the G0G1 peak of chicken erythrocytes), documented the former to be small, with S.D. values varying from 0.0 to 0.016, while the latter were considerably higher with S.D. values varying from 0.077 to 0.123. Since the intraday variation of the DNA ratio was consistently low and the interday variation strongly correlated to the position of the red fluorescence test beads, it was possible to minimize the interday variation of the DNA ratio, by calculating the DNA index as the ratio between the DNA ratio of the sample and that of an external control (buffy coat leukocytes). Analyzing normal bone marrow and calculating the DNA index (DI) on the basis of these ratios, the confidence limits of the DI were decreased by more than half the values obtained when DI calculation was based solely on an internal standard, thereby making subsequent ploidy determinations of patient samples more precise. We conclude that this setup of internal and external standards allows accurate determinations of DNA aneuploidy even in an assay where whole cells labelled for surface antigen and DNA content are analyzed.

Hybrid acute leukemia: therapeutical implications of immunological phenotyping.

We phenotyped blood and bone marrow cells from a patient with acute Ph1+ acute leukemia longitudinally during the four months he received intensive chemotherapy. At presentation this case of biphenotypic acute leukemia had two immunologically different types of blast cells, one expressed CD10 (CALLA), CD13 (MY7) and CD33 (MY9) but lacked CD20 (B1), the other type expressed no CD10 or CD33. The phenotype, during AML induction therapy, changed to a more CD10+, CD20+ ALL one. ALL therapy based on these findings induced improvement in bone marrow function but the patient died of septicemia at day 134. The use of concomitant immunophenotyping (IP) and cell cycle analysis had shown proliferation advantage of the more lymphoid malignant cells. These results suggest that it is possible to induce lineage-associated changes in the phenotype of hybrid malignant cells and that these leukemias might be treated best according to longitudinal immunophenotyping of the blast cells.

Evolution of Ig- and T-cell receptor gene configuration in a Ph1+ hybrid leukemia patient.

In a longitudinal study of a 32-year-old male with Ph1+ hybrid leukemia we have followed the immunophenotype and configuration of Ig- and TCR genes during the course of different chemotherapy regimens directed first against the myeloid and later against the lymphoid components of the disease. We identified changes in all parameters, interpretable as an evolution of the malignant clone resulting in a leukemic switch towards a more lymphoid character. Thus, while the expression of the myeloid antigens CD13 and CD33 decreased, that of CD10 (CALLA) and CD20 (B1) increased. Moreover, while the configuration of the Ig heavy and light chain lambda genes remained constant during the whole period of treatment, that of the Ig light chain kappa gene and TCR beta gene displayed extensive rearrangements after initiation of ALL therapy. Since this patient represents a de novo acute leukemia as evaluated by location of the translocation-breakpoint on chromosome 22, our data clearly indicate that Ig- and TCR gene rearrangements might prove a valuable addition in monitoring Ph1+ hybrid leukemias, providing guidelines for optimizing chemotherapy.

Monoclonal antibodies in myeloid diseases: prognostic use in acute myeloid leukaemia.

Bone marrow cells from 109 patients (median age 60) with newly diagnosed acute myeloid leukaemia (AML) were prospectively immunophenotyped (IP) and the prognostic value of monoclonal antibody (MAB) reactivities was analysed to detect differences in complete remission rates and survival, not only between groups of MAB + and - bone marrow cells, but also in cases with or without prominent MAB reactivity as compared to normal BM reactivity of the respective MABs. This approach was based on the assumption that the qualitative expression of antigens is not an all or none phenomenon, but that different degrees of expression of antigens exist. Patients with significantly elevated CD13 (MY7+) cells in bone marrows (CD13 greater than reference value + one standard deviation) (S.D.) showed decreased probability of entering CR (p less than 0.05) and a significantly shorter survival (p less than 0.05). Superior CR rates (p less than 0.05) without difference in long-term survival were seen in patients with low CD33 (MY9) or low HLA-DR expression, while high CD14 (MY4) expression showed a trend towards an adverse factor (p = 0.12). No other antibody reactivities showed differences in CR rates (CD3, CD20, CDw65 (VIM-2) and NAT-9). The more prominent bone marrow expression of CD33 antigen than CD13 (CD33/CD13 greater than 1) correlated to a better chance of entering CR (p = 0.01) and to improved survival (p = 0.002), while the expression of high numbers of VIM-2+ cells was a favourable prognostic factor regarding length of survival (p = 0.002). The importance of a high CD33/CD13 ratio as a positive prognostic factor was evaluated using stratified analysis according to age or leucocyte counts at presentation. In both cases, CD33/CD13 was associated with longer survival (age: p = 0.05, leucocyte counts: p = 0.03). A Cox multiparameter analysis revealed that the CD33/CD13 ratio was a favourable prognostic factor (p = 0.03) together with age (p = 0.001) and leucocyte counts in peripheral blood (p less than 0.01). We conclude that establishing the immunologic phenotype can be of prognostic value in cases of AML, especially with regard to the relationship between the CD33 and CD13 antigens.

Family studies of hereditary hemochromatosis in Denmark and the Faroe Islands.

Pedigree studies were performed based on one Faroese and four Danish probands with overt idiopathic hemochromatosis (IH). The study consisted of HLA typing and determination of biochemical iron status indicators (serum transferrin saturation, serum ferritin). In total, 130 persons were evaluated. The screening identified 6 homozygous (h/h) subjects with preclinical IH, 46 heterozygous (h/n), and 8 normal (n/n) subjects, while 39 subjects were classified as normal or heterozygous (n/h?). One family demonstrated both a homozygous x heterozygous as well as a heterozygous x heterozygous mating. Recombination between the HLA region and IH locus occurred possibly in three subjects in three different families. The significance of detailed screening in families with probands with IH is discussed.

Monoclonal antibody ratios in malignant myeloid diseases: diagnostic and prognostic use in myelodysplastic syndromes.

Based on a 6 1/2-year study of bone marrows (BM) from 74 consecutive adult patients with newly diagnosed primary myelodysplastic syndromes (MDS), we have evaluated the role of immunophenotyping (IP) for subclassification purposes with a selected panel of monoclonal antibodies (Mab). Compared to normal BM IP revealed increased numbers of Mab-defined immature myeloid cells (defined by Mab MY7 [CD13] and MY9 [CD33] (P less than 0.05). Furthermore, decreased numbers of mature myeloid cells (defined by Mab NAT-9 II:3F-6F [NAT-9]) (P less than 0.001) were demonstrated in all French-American-British (FAB) subtypes except refractory anaemia with sideroblasts (RA-S). Since expression of CD13 and CD33 antigens on immature myeloid cells is variable, IP based on a single Mab was often found to be non-informative. However, the construction of myeloid antibody ratios (MAR), designed to give higher figures with increasing numbers of immature myeloid cells and/or decreasing numbers of mature myeloid cells in the BM, disclosed increasing mean ratio values with progressing FAB subtypes. More significant however, was that different prognostic subgroups based on the MAR could be identified independently of the FAB classification. We conclude that the use of IP--especially when MAR is included--is useful in prognostic scoring systems in MDS.