Proliferative activity of leukaemic blasts and cytosine arabinoside pharmacodynamics are associated with cytogenetically defined prognostic subgroups in acute myeloid leukaemia.

The biological mechanisms responsible for the association of specific karyotypes with prognosis in acute myeloid leukaemia (AML) remain largely unclear. A prospective study was performed to evaluate how far cytogenetically defined prognostic subgroups of AML differ in their proliferative activity as a potential mechanism for differential sensitivities to S-phase-specific induction chemotherapy comprising cytosine arabinoside (AraC). One hundred and eighty-seven patients with de novo AML were included in the study; 25 patients with a favourable [inv(16), t(8;21), t(15;17)] karyotype, 99 with a normal karyotype, 29 with an unfavourable karyotype (-5, 5q-, -7, 7q-, complex aberrations) and 34 with cytogenetic aberrations of unknown prognostic significance (all others). The favourable group demonstrated the highest ex vivo proliferative activity (PA) (3.41 pmol/105 cells), significantly (P = 0.02) exceeding the unfavourable group with the lowest PA (0.72) and the group with a normal karyotype (1.06) or with karyotype of unknown significance (1.05) that both demonstrated an intermediate PA. Samples with a high PA (> median of the whole group) were more likely to produce interleukin 3, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) (56%, 43% and 50%) than cells with a low PA (33%, 36% and 36%; n.s.). The effect of priming by exogenous GM-CSF or G-CSF was significantly more pronounced in samples with a low PA than in rapidly proliferating samples (P < 0.01). For the whole group, a high PA was closely associated with an increased incorporation of AraC triphosphate (AraCTP) into the DNA (P < 0.0001). Clinically, a high PA was associated with a better complete remission (CR) rate in the normal (95% versus 62%) and the unfavourable group (75% versus 33%). The significant differences in proliferative activity between cytogenetic subgroups of AML are associated with increased cytosine arabinoside pharmacodynamics and constitute one potential mechanism for the different response of cytogenetic subgroups to AraC-based induction therapy.

Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca(2+) release studies in bronchial epithelial cells.

P2Y(2) receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl(-) secretion via an increase in [Ca(2+)](i) (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl(-) secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap(4)A). In human and rat lung membranes, Ap(4)A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap(4)A caused an increase in [Ca(2+)](i) with an EC(50) of 17 microM in human bronchial epithelial cells (HBE1). The [Ca(2+)](i) rise evoked by ATP and UTP was completely, but that induced by Ap(4)A only partially, caused by release of Ca(2+) from internal stores. Moreover, the potency of Ap(4)A to mobilize Ca(2+) was lower than that of ATP and UTP (EC(50) 1.5 and 1.8 microM, respectively), and the maximal increase in [Ca(2+)](i) was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap(n)A analogues tested (n = 3 to 6) caused a comparable [Ca(2+)](i) increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca(2+)](i) found after a second pulse of either UTP or Ap(4)A. Although specific binding characteristics and functional responses of Ap(4)A on lung cells are in favor of a distinct receptor for Ap(4)A, the cross-talk between UTP and Ap(4)A in HBE1 cells and the only slight differences in Ca(2+) mobilization by ATP or UTP and Ap(4)A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap(4)A activates one of the nucleotide receptors already described.

Cytogenetic subgroups in acute myeloid leukemia differ in proliferative activity and response to GM-CSF.

The current study was undertaken to search for differences in the biology of cytogenetic subgroups in patients with de novo acute myeloid leukemia (AML). In addition, factors influencing the metabolism of cytosine arabinoside (araC) as the key agent of antileukemic activity were assessed. Bone marrow aspirates from 91 patients with newly diagnosed AML in whom karyotypes were successfully obtained were analyzed: (1) for spontaneous proliferative activity by 3H-thymidine (3H-TdR) incorporation; (2) proliferative response to GM-CSF by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4-h exposure to 3H-TdR (0.5 microCi/ml); and (3) parameters of araC metabolism comprising 3H-araC uptake in vitro and the activities of polymerase alpha (poly alpha), deoxycytidine kinase (DCK) and deoxycytidine deaminase (DCD). According to the results of chromosome analyses four cytogenetic subgroups were discriminated: (I) normal karyotypes (n = 38); (II) favorable karyotypes [t8;21), t(15;17), inv(16)] (n = 16); (III) unfavorable karyotypes [inv (3), -5, 5q-, t(6;9), +8, t (9;11), complex abnormalities] (n = 20); (IV) karyotypes of unknown prognostic significance (n = 17). Proliferative activity of leukemic blasts was significantly higher in favorable karyotypes (group II) as compared to cases with unfavorable cytogenetics (group III) with median values and range for 3H-TdR uptake in group II of 2.48 pmol/10(5) cells (0.28-25.8) and in group III of 0.51 pmol/10(5) cells (0.04-7.6) (P = 0.0096). The respective values in group I and group IV were 0.7pmol/10(5) cells (0.0-6.7) and 0.98 pmol/10(5) cells (0.0-4.0), respectively. Inversely, response to GM-CSF, as defined by an increase in 3H-TdR incorporation >1.5- fold over control values after 48h of GM-CSF exposure, was significantly lower for patients with a favorable karyotype (group II) as compared to group I (P = 0.04) and group III (P = 0.013). No significant differences between karyotype groups I, II, III and IV were found for 3H-araC incorporation, nor for the activities of poly alpha, DCK and DCD. These data demonstrate differences in the biology of cytogenetic subgroups in AML which may partly explain the well established differences in clinical outcome.

The pharmacodynamic basis for the increased antileukaemic efficacy of cytosine arabinoside-based treatment regimens in acute myeloid leukaemia with a high proliferative activity.

The current study was initiated to explore the mechanisms underlying the previously demonstrated association between the proliferative activity of leukaemic blasts and the response to cytosine arabinoside (AraC)-based therapy in de novo acute myeloid leukaemia (AML). The activity of key enzymes of AraC metabolism-deoxycytidine kinase (DCK), cytidine deaminase (DCD) and polymerase alpha (PolyA) were determined in blast cells from 33 patients. In addition, formation and retention of intracellular levels of AraC triphosphate (AraCTP) and DNA incorporation of AraC were measured, as was the proliferative activity of leukaemic blasts by [3H]-TdR incorporation before and after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) or granulocyte CSF (G-CSF) for 48 h. AraC incorporation into the DNA (median 0.60 pmol/105 cells) was significantly related to the proliferative activity of AML blasts (r = 0.74, P < 0.001). Similarly, priming with GM-CSF or G-CSF increased both the proliferative activity of AML blasts by a median of 1.84- and 1.64-fold, respectively, and the incorporation of AraC into the DNA (1.29- and 1.40-fold respectively). In contrast, no relationship was found between the endogenous proliferative activity (EPA) and enzyme activities regulating AraC activation (DCK; median 4.70 pmol/min/mg protein), inactivation (DCD; median 2.92 pmol/min/mg protein) or inhibitory effects (PolyA; median 1.50 pmol/min/mg protein), nor the formation or retention of AraCTP (median 306.1 ng/107 cell and 1.6 h respectively). When samples were grouped according to EPA (more than or less than the median), slowly proliferating specimens had a higher response to cytokine priming for proliferative activity and incorporation of AraC into DNA. Clinical data of 15 patients were available. Although all eight patients with a high endogenous proliferative activity reached complete remission, only four out of seven patients with a low proliferative activity responded, whereas the other three patients were non-responders (P = 0.077).

Successful modulation of high-dose cytosine arabinoside metabolism in acute myeloid leukaemia by haematopoietic growth factors: no effect of ribonucleotide reductase inhibitors fludarabine and gemcitabine.

High-dose cytosine arabinoside (AraC)-containing regimens have shown the highest antileukaemic efficacy of all currently used regimens in the treatment of acute myeloid leukaemia (AML). This study aimed at increasing the antileukaemic potential of high-dose AraC by raising intracellular levels of AraC triphosphate (AraCTP), which is the mediator of cytotoxicity, via biochemical modulation by inhibitors of ribonucleotide reductase (RR) or haematopoietic growth factors (HGFs). Blasts from patients with de novo AML were analysed for their formation of AraCTP under high-dose AraC conditions (20 microM over 3 h) without prior modulation (n = 47) after a 2-h pre-exposure with fludarabine (50 microg/ml) (n = 40) or gemcitabine (30 ng/ml) (n = 40) and after a 48-h pre-exposure to granulocyte colony-stimulating-factor (G-CSF; 100 ng/ml) (n = 27) or granulocyte-macrophage colony-stimulating-factor (GM-CSF; 100 U/ml) (n = 28). Unmodulated formation of AraCTP (median 239.8 ng/107 cells) could not be increased via modulation by gemcitabine (232.4 ng/107 cells) or fludarabine (247.8 ng/107 cells). The lack of effect of RR inhibitors was also observed for all other known metabolites of AraC [Ara-cytosine monophosphate (CMP), Ara-cytosine diphosphate (CDP), AraCDP-choline, Ara-uridine monophosphate (UMP), Ara-uridine diphosphate (UDP) and Ara-uridine triphosphate (UTP)]. In contrast, pre-exposure to HGFs led to significant increases in AraCTP formation (G-CSF 556.0 ng/107 cells, 2.31-fold increase, P < 0.001; GM-CSF 447.9 ng/107 cells, 1.87-fold increase, P < 0.0001). To establish the mechanism responsible for these effects, the activity of the rate-limiting enzyme of AraC metabolism, deoxycytidine kinase (dCK), was investigated (n = 33). In vivo exposure to GM-CSF led to increases in dCK activity from unmodulated values at 0 h (29.8 pmol/min/mg protein) to 34.3 pmol/min/mg protein at 24 h (1.15-fold increase) and 54.5 pmol/min/mg protein at 48 h (1. 83-fold increase). The raise in dCK activity over 48 h was significant (P < 0.013).

In human and rat lung membranes [35S]GTPgammaS binding is a tool for pharmacological characterization of G protein-coupled dinucleotide receptors.

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.

Intensive therapy for high-risk myelodysplastic syndromes and the biological significance of karyotype abnormalities.

Therapy for myelodysplastic syndromes (MDS) has been less than effective when based on low-dose treatment or supportive measures only, including hematopoietic growth factors. Recently, based on the percentage of bone marrow blasts, the number of cytopenic cell lines and cytogenetics, clinical risk groups have been defined more precisely. Recent studies applying intensive acute myeloid leukemia (AML)-type therapy to high-risk MDS have produced remissions ranging from 45 to 79%. Advances in the understanding of the biology of MDS clearly point to cytogenetics rather than morphologic subtype as being of prognostic relevance. Hence, new treatments need to be developed for patients with unfavorable karyotypes and complex abnormalities in particular. These MDS subtypes are characterized by low spontaneous proliferative activity and low autocrine production of hematopoietic growth factors. The subtypes are, however, highly sensitive to external stimulation by granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF). New therapies could emerge from these findings, for example, priming high-risk MDS patients with hematopoietic growth factors in combination with intensive AML-type treatment. Recent studies suggest that incorporating high-dose AraC into an intensive drug combination could further improve the outcome of high-risk MDS.

Plasmacytosis in acute myeloid leukemia: two cases of plasmacytosis and increased IL-6 production in the AML blast cells.

An increased plasma cell count in the bone marrow occurs in a subgroup of patients with acute myeloid leukemia (AML). The pathogenic mechanism for this plasmacytosis is unclear. In this report we describe two patients with AML and plasmacytosis who shared some features of their diseases. The morphological subtypes were AML M4 and M4eo; the leukemias were secondary to cytotoxic pretreatment, and complex cytogenetic changes were found in the leukemic cells of both patients. There was a marked increase in the number of bone marrow plasma cells in both cases and no monoclonal immunoglobulin was detectable. The IgH-CDR3 gene scan depicted a monoclonal IgH rearrangement in the bone marrow cells of one patient. Analysis of the cytokine production of the leukemic cells showed a high production of IL-6 of the leukemic blast cells in the in vitro cell culture and a high cytoplasmic IL-6 in the leukemic cells as revealed by immunocytology. We describe the clinical picture of a type of secondary AML with FAB M4 morphology associated with bone marrow plasmacytosis. We suggest that paracrine growth stimulation of plasma cells by paraneoplastic IL-6 production of the leukemic blast cells contributes to the plasmacytosis observed in patients with AML.

Renin in acute myeloid leukaemia blasts.

Hypokalaemia is a clinical phenomenon in patients with acute myeloid leukaemia (AML) to which activation of the renin-angiotensin system (RAS) may contribute. Recently monocytes were found to express renin, the key initializing enzyme of the RAS. By RT/PCR, transcripts for renin were detected in four of 18 bone marrow samples from patients with AML. Three leukaemic cell lines, isolated monocytes, bone marrow stromal cells and 25 peripheral blood and 24 bone marrow samples of normal controls were negative for renin transcripts. In view of the importance of local RAS in other tissues, the expression of renin in the bone marrow of AML patients warrants further investigation.

First clinical evaluation of the CELL-DYN 3200 haematology analyser.

A prototype of the CELL-DYN 3200 haematology analyser was evaluated in a tertiary care hospital laboratory. Precision, effects of sample ageing, linearity, carry-over, and comparability of cellular blood counts and five-part leucocyte differentiation were determined in accordance with the ICSH guidelines for the evaluation of blood cell analysers; the results were satisfactory for all parameters tested: haemoglobin concentration, RBC, MCV, WBC, platelet count, and counts of neutrophils, lymphocytes, monocytes, and eosinophils. Two-hundred and forty-seven routine blood samples were used for the comparability studies. The cellular blood count results from the CELL-DYN 3200 and the Bayer Diagnostic H-1 systems corresponded closely (correlation coefficient r > 0.96 for all parameters). For 201 samples without an instrument-generated suspect flag the same was true with regard to the differential parameters, although somewhat lower correlation was observed for monocyte counts (r = 0.88). Comparisons to 400-cell microscopic differentials gave similar results (r > 0.93 for neutrophil, lymphocyte and eosinophil counts). Our results suggest that the CELL-DYN 3200 analyser will serve the needs for automated blood cell counting and differential leucocyte counting in a tertiary care hospital laboratory.

Activity of thymidine kinase and of polymerase alpha as well as activity and gene expression of deoxycytidine deaminase in leukemic blasts are correlated with clinical response in the setting of granulocyte-macrophage colony-stimulating factor-based priming before and during TAD-9 induction therapy in acute myeloid leukemia.

The present study was undertaken to assess the predictive value of pretherapeutic determinants of ara-C metabolism and proliferative activity of leukemic blasts for early response to antileukemic therapy in the setting of granulocyte-macrophage colony-stimulating factor (GM-CSF)-based priming before and during TAD-9 induction in 36 consecutive patients with de novo acute myeloid leukemia (AML). Ara-C metabolism was assessed by the activities of deoxycytidine kinase (DCK), deoxycytidine deaminase (DCD), DNA polymerase alpha (Poly alpha), and overall polymerase (overall Poly). The fraction of cells in S phase (%S phase) and thymidine kinase (TK) activity were determined as a measure of proliferative activity. Early response to therapy was defined by the percentage of leukemic blasts in the bone marrow 5 to 7 days after completion of TAD-9 with less than 5% signaling an adequate response and greater than 5% indicating an inadequate early reduction, respectively. While neither %S phase, DCK, nor overall Poly activity were predictive for early response, TK and Poly alpha activities were significantly higher for cases with adequate blast cell clearance. The respective median values were for TK 3.8 versus 1.85 pmol/min/mg protein (P = .012), and for Poly alpha 1.9 versus 0.69 pmol/min/mg protein (P = .014). An inverse relation was detected for DCD activity which was significantly lower in responding patients with a median of 0.33 nmol/min/mg protein (range, 0.0 to 29.5) as compared to a median of 5.1 nmol/min/mg protein (range, 0.11 to 8.45) in early nonresponders, (P = .009). Taking the respective median values as arbitrary cut-points for high or low enzyme activities, responders and nonresponders could be discriminated prospectively. Hence, 14 of 16 cases (88%) with DCD activities below the median of 1.56 nmol/min/mg protein responded as compared to only 3 of 14 (22%) patients with higher DCD activities (P = .0004). From the 15 patients with TK activity above the overall median of 3.2 pmol/min/mg protein, 11 cases (73%) achieved an adequate blast cell clearance while only 6 of 17 cases (35%) with lower values responded (P = .035). Similarly, 12 of 15 patients (80%) with high Poly alpha levels (>1.22 pmol/min/mg protein) responded to induction therapy as compared to only 5 of 14 patients (36%) with lower enzyme activities (P = .02). By logistic regression analysis of enzyme activities, DCD activity was found to be the most sensitive parameter to predict an adequate blast cell clearance (P = .032). Activities of DCD and TK were not only associated with initial response but were also found predictive for remission duration. Hence, from 11 patients with low TK levels 8 (73%) relapsed within 1 year, whereas only 2 of 11 (18%) patients with high TK activity experienced a recurrence of their disease (P = .015). Six of 9 (66%) patients with higher than median DCD levels relapsed within 1 year, whereas 10 of 14 patients (71%) with lower DCD levels had a longer remission duration (P = .085). Analysis of DCD gene expression at the mRNA level by a semi-quantitative reverse transcriptase-polymerase chain reaction method showed that a high transcription rate of the DCD gene was associated with high enzyme activities and vice versa. Hence, the observed intraindividual differences in DCD activity are a reflection of differences in gene activity and transcription rate rather than of variants in translation. Although further analyses are needed to elucidate the molecular mechanisms that determine the variation of enzyme activities in individual patients, the present study strongly suggests that pretherapeutic determination of TK and Poly alpha as well as of DCD allows to predict response to TAD-9 + GM-CSF induction therapy and may provide the means for the development of a risk adapted treatment strategy.

Paraneoplastic hypokalemia in acute myeloid leukemia: a case of renin activity in AML blast cells.

Hypokalemia due to renal potassium loss has frequently been observed in patients with acute myeloid leukemia (AML). The pathogenic mechanism for this hyperkaluresis is unclear. In this report we describe a patient with AML FAB M4, in whom the clinical course, the electrolyte disturbances, the serum aldosterone levels, and the diffuse hyperplasia of the adrenal cortex documented a typical case of marked secondary hyperaldosteronism. On analysis of the leukemic cells of this patient compared with normal bone marrow cells, a significant increase of renin-like activity in the cytosol of the blast cells was noted. Activation of the renin-angiotensin-aldosterone system by paraneoplastic production of renin-like activity in AML blast cells might contribute to the hypokalemia often observed in patients with acute myeloid leukemia.

Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia.

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)