Cytogenetic subgroups in acute myeloid leukemia differ in ... - Nature

Leukemia (2001) 15, 377–384  2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu

Cytogenetic subgroups in acute myeloid leukemia differ in proliferative activity and response to GM-CSF G Jahns-Streubel1, J Braess1, C Schoch1, C Fonatsch2, D Haase3, C Binder3, B Wo¨rmann3, T Bu¨chner4 and W Hiddemann1 1

Department of Medicine III, University Hospital Großhadern, Ludwig Maximilians University, Munich; 3Department of Hematology and Oncology, Georg-August-University, Go¨ttingen; 4Department of Hematology and Oncology, Westfa¨lische Wilhelms University, Mu¨nster, Germany; and 2Department of Medical Biology, University of Vienna, Austria

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 ␮Ci/ml); and (3) parameters of araC metabolism comprising 3H-araC uptake in vitro and the activities of polymerase alpha (poly ␣), 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/105 cells (0.28– 25.8) and in group III of 0.51 pmol/105 cells (0.04–7.6) (P = 0.0096). The respective values in group I and group IV were 0.7 pmol/105 cells (0.0–6.7) and 0.98 pmol/105 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 48 h 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 ␣, 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. Leukemia (2001) 15, 377–384. Keywords: cytogenetics; acute myeloid leukemia; proliferative activity; growth factors

Introduction With the development of intensive induction chemotherapy and the improvement of supportive care, complete remissions (CR) are nowadays achieved in 50% to 80% of adult patients with acute myeloid leukemia (AML), but only 20% to 30% of cases experience long-term disease-free survival and potential cure.1–4 Increasing insights into the biology of AML indicate that distinct cytogenetic and molecular aberrations can be identified that are closely related to treatment outcome. Cytogenetic analyses thus allow discrimination of subgroups of patients with favorable, unfavorable and intermediate prog-

Correspondence: W Hiddemann, Department of Medicine III, University Hospital Gro␤hadern, Ludwig Maximilians University, Marchioninistr 15, 81377 Mu¨nchen, Germany; Fax: 89 7095 8875 Received 3 April 2000; accepted 29 September 2000

nosis and provide the means to develop risk-adapted treatment strategies.5–9 New therapeutic approaches that have been investigated recently include the application of cytosine arabinoside (araC) at high doses10,11 and preliminary data indicate substantial differences in the efficacy of this treatment between distinct cytogenetic subgroups.12–17 High expectations have also been associated with the priming of leukemic blasts by hematopoietic growth factors to enhance their sensitivity for subsequent chemotherapy. Although this approach was supported by promising in vitro data,18–22 the available data from prospective clinical studies are mostly disappointing.23–30 Such a global conclusion, however, appears too simplistic and neglects potential differences in the efficacy of priming concepts for distinct subtypes of AML. In particular, no data are yet available to relate the use of hematopoietic growth factors to pretherapeutic determinants of prognostic or biologic relevance such as cytogenetics. Overall, more information about the biology of AML, the determinants of response to cytostatic drugs, such as araC, and the mechanisms underlying the prognostic significance of cytogenetic subgroups is needed to develop appropriate regimens for patients facing different long-term expectations. A variety of biologic characteristics of leukemic blasts have been investigated with regard to their predictive value for treatment response. Analyses about the major determinants of araC metabolism in leukemic cells led to different conclusions about their relevance to treatment outcome. While some studies found DCK activity in AML blasts to be predictive for therapeutic response31,32 other groups suggested that some still unidentified mechanism of metabolic control rather than the amount of active enzyme determines the level of araC incorporation and cytotoxicity.33,34 The same controversy also relates to DCD activity. A recent investigation by our group35 confirmed earlier studies and found a high DCD activity to be predictive for resistance to therapy31,36 while this association was rejected by other authors.37,38 Conflicting data have similarly been reported about the association between the proliferative activity of leukemic blasts and clinical outcome.39–41 Lo¨wenberg et al42 described an inverse relation between proliferative activity of leukemic blasts and long-term prognosis while our group could demonstrate that patients with a high proliferative activity of AML blasts in vitro have a higher probability to experience an adequate response to induction therapy as compared to cases with a low spontaneous proliferation rate.43 Sensitivity of blast cells to GM-CSF in vitro appeared to be inversely related with early treatment response which may be explained by the autocrine production of hematopoietic growth factors in AML blasts.44–47 These results were confirmed by Tsuzuki and coworkers,48 showing that patients whose leukemic cells had a positive proliferative response to growth factors had a poorer outcome. In all these studies the only generally accepted determinant

Cytogenetic subgroups in AML G Jahns-Strenbel et al

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of prognosis, namely cytogenetics, has so far been disregarded. In children with acute lymphoblastic leukemia, on the other hand, an association between high numerical chromosome aberrations as revealed by a high DNA index and enzyme activities involved in the metabolism of methotrexate and treatment response was observed.49,50 In the current study cytogenetic analysis provided the basis to assess the clinical relevance of additional factors including parameters of intracellular araC metabolism, blast cell proliferative activity and response to GM-CSF stimulation. Parameters of araC metabolism comprised the 3H araC uptake into DNA of blast cells in vitro and the activities of poly ␣, deoxycytidine deaminase (DCD) and deoxycytidine kinase (DCK). The proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation into the DNA while the sensitivity to growth factor stimulation was defined as an increase in 3H-TdR incorporation ⬎1.5fold over control values after GM-CSF exposure in vitro. This multifactorial approach attempts to combine the major determinants of treatment response in order to gain a better understanding of their complex interrelation and their relevance for the design of biology-based, risk-adapted or even risk-modulating treatment strategies.

as the period from fulfillment of CR criteria to relapse. Patients, who died in first CR or underwent bone marrow transplantation in first CR were censored at the respective time points. Survival was calculated from the first day of chemotherapy to death.

Patients, materials and methods

Chromosome analyses

Patients and treatment protocols

Chromosome analyses were performed on short-term cultures of bone marrow cells. Cells were incubated for 24 h at 37°C in RPMI supplemented with 20% FCS, 100 U /ml recombinant human (rh) granulocyte colony-stimulating factor (Amgen, Thousand Oaks, CA, USA), 100 U/ml rh granulocyte–macrophage colony-stimulating factor (Behringwerke, Marburg, Germany), 100 U/ml interleukin-3 (Behringwerke), 1 U/ml erythropoietin (Boehringer, Mannheim, Germany) and 50 ng/ml stem cell factor (Genzyme, Boston, MA, USA). Chromosome preparation and modified GAG-SSC staining was performed as previously described.53,54 The karyotypes were classified according to the International System of Chromosome Nomenclature, (ISCN 1995).55 In addition, the following four prognostic groups were defined: (I) normal karyotype; (II) favourable karyotypes *t8;21), t(15;17), inv(16)]; (III) unfavorable karyotypes *inv (3), −5, 5q−, t(6;9), +8, t (9;11), complex abnormalities (three or more abnormalities present)]; (IV) karyotypes of unknown prognostic significance.

Patients presenting with newly diagnosed AML and a bone marrow blast infiltration of 70% or more were eligible for the current investigation. The diagnosis of AML was based on FAB criteria and complementary cytochemical and immunological analyses. All patients were treated according to the protocols of the German AML Cooperative Group (AML-CG 91). Eligible patients underwent the TAD-9 regimen as first induction cycle.51 TAD-9 comprized 100 mg/m2/day araC given by contious i.v. infusion on days 1 and 2 followed by short-term infusions of araC 100 mg/m2 every 12 h on days 3–8; 6-thioguanine 100 mg/m2 every 12 h orally on days 3–9 and 60min infusions of daunorubicin 60 mg/m2/day on days 3–5. Patients ⬍60 years of age subsequently underwent a second induction cycle comprising either TAD-9 again or HAM consisting of high-dose araC (3.0 g/m2 every 12 h by i.v. infusion on days 1–3) and mitoxantrone (10 mg/m2/day by i.v. infusion on days 3–5). Patients above the age of 60 years were only treated with a second TAD-9 cycle if they revealed persistent blasts in the bone marrow at day 16.16,52 After attaining a complete remission all patients received one additional course of TAD-9 for consolidation which was followed by monthly 5-day maintenance using rotating courses of AD/AT/AC/AT/AD etc for 3 years. AD comprised araC (100 mg/m2 every 12 h s.c. days 1–5) and daunorubicin (45 mg/m2/day 60 min i.v. infusion on days 3 and 4); AT consisted of araC (100 mg/m2 every 12 h s.c. days 1–5) and 6thioguanine (100 mg/m2 every 12 h orally on days 1–5); AC comprises araC (100 mg/m2 every 12 h s.c. days 1–5) and cyclophoshamide (1 g/m2 i.v. on day 3).51 Complete remission (CR) was defined as the absence of leukemia-associated symptoms, a bone marrow of normal cellularity with less than 5% blast cells and normal peripheral blood counts with more than 1500 granulocytes per cubic millimeter and more than 100 000 platelets per cubic millimeter, and the absence of circulating blasts and extramedullary leukemic manifestations. Remission duration was defined Leukemia

Study conduct Prior to its initiation, the study was approved by the local ethic committee of the participating institutions. It strictly adhered to the updated version of the Helsinki Declaration. All patients gave their informed consent to participate in the current evaluation after having been informed about ist purpose and investigational nature.

Cell processing Samples were taken by bone marrow aspiration and were subsequently subjected to Ficoll–Hypaque density gradient separation before further processing.

Incubation procedure Blast cells were cultured for 48 h in the presence or absence of GM-CSF (100 U/ml) kindly provided by Behringwerke at a concentration of 1 × 106 cells in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% heat inactivated fetal calf serum (FCS), glutamine (300 ␮g/ml) and antibiotics (penicillin 60 ␮g/ml; streptomycine 133 ␮g/ml) at 37°C in a 5% CO2 atmosphere. The concentration of GM-CSF used in these assays reflects the concentration of the growth factor that can be achieved clinically. 3

H-TdR incorporation into DNA

After preincubation in the presence or absence of GM-CSF, 4 × 105 AML blasts were plated in 24-well microtiter plates of a final volume of 2 ml IMDM with 10% heat inactivated FCS, glutamine and antibiotics and 3H-TdR (0.5 ␮Ci/ml,


5 Ci/mmol; Amersham Buchler, Braunschweig, Germany) was added for an additional 4 h. Cells were harvested on DE81 filter paper (Whatman, Maidstone, UK), washed twice with 0.5 m sodium phosphate buffer (pH 7.0) and H2O, and once with 96% ethanol. Sensitivity to GM-CSF was defined by an increase in 3H-TdR incorporation ⬎1.5-fold over control values after the 48 h GM-CSF exposure. All experiments were performed in triplicate.

3

H-ara-C incorporation into DNA

After preincubation 8 × 105 AML blasts were plated in 24-well microtiter plates at a final volume of 2 ml IMDM with 10% heat inactivated FCS, glutamine and antibiotics. 3H-ara-C, (1 ␮Ci/ml; 31 Ci/mmol; Amersham Buchler) and increasing amounts of unlabeled araC to 0.03 and 0.13 ␮m final concentrations were added for an additional 4 h. Cells were harvested on DE81 filter paper (Whatman), washed twice with 0.5 m sodium phosphate buffer (pH 7.0) and H2O, and once with 96% ethanol in order to bind radiolabeled DNA as previously described.56 All experiments were performed in triplicate.

Cell extract preparations Blast cells were washed in 0.9% NaCl and then used for the determination of enzyme activities. For preparation of cell extracts, 107 blast cells were suspended in 100 ␮l 50 mm TrisHCl, pH 7.4. Blast cells were lysed by the freeze–thawn procedure which was repeated three times. Cellular debris and unsolved proteins were pelleted by centrifugation at 12 000 g for 15 min at 4°C. The supernatant was assayed for enzyme activities and protein concentration.57

Production and purification of the monoclonal antibody (Mab) SJK 237–71 against DNA polymerase ␣ The hybridoma cell line SJK 237–71 was obtained from the American Type Culture Collection, Rockville, MD, USA. As previously described in detail, SJK 237–71 hybridoma cells secrete monoclonal IgG 1 antibodies which react with polymerase ␣ without exhibiting neutralizing activity.58 Hybridoma cells were cultivated in minimal essential medium (MEM) supplemented with 10% heat inactivated FCS, glutamine (300 ␮g/ml) and antibiotics (penicillin 60 ␮g/ml; streptomycin 133 ␮g/ml) at 37°C in a 5% CO2 atmosphere. Monoclonal antibodies were isolated from the cell culture supernatants using the commercial Affi-Gel Protein A MAPS II (monoclonal antibody purification system) kit (BioRad, Munich, Germany) according to the manufacture’s instructions. The concentration of DNA polymerase alpha-Mab was assessed by ELISA, using as coating antibody 31500045 (Jackson ImmunoResearch, Baltimore, MD, USA), as detection antibody peroxidase conjugated 315035003 (Jackson), ophenylendiamine dihyrochloride (OPD) (Jackson) as substrate and mouse IgG (Sigma, Deisenhofen, Germany) as internal standard.

DNA polymerase ␣ assay

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DNase activated calf thymus DNA which served as a starter of the polymerase ␣ assay was prepared as described by Aposhian and Kornberg.59 The DNA polymerase alpha assay was performed according to Hammond et al,60 with the following modifications. 20 ␮l cell extract was incubated with 4 mg protein A-sepharose (Pharmacia, Freiburg, Germany), and 25 ␮l of purified MAB SJK 237–71 (0.1 mg/ml) in phosphate-buffered saline (PBS) for 2 h at 4°C. After washing of the immobilized immunocomplexes with 1 ml PBS and 1 ml reaction buffer (5 mm MgCl2, 1 ml dithiothreitol (DTT) and 50 mm Tris/HCl pH 7.4), a final volume of 100 ␮l was added, containing 30 ␮g DNase-activated DNA, 10 ␮m TTP, 10 ␮m dCTP, 10 ␮m dATP, 10 ␮m dGTP, 5 mm MgCl2, 1 mm DTT, 50 mm Tris-HCl pH 7.4 and 1␮Ci dCTP (26 Ci/mmol; Amersham Buchler). The reactions were incubated for 15 min at 37°C. Incorporation of 3H-dCMP into DNA was determined by using DE81 filter paper (Whatman) as previously described.35 All experiments were performed in triplicate.

Thymidine and deoxycytidine kinase assays For thymidine kinase (TK) and deoxycytidine kinase (DCK) assays61 100 ␮l reaction buffer (final concentrations: 100 mm Tris-HCl pH 8.0, 4.5 mm ATP, 5 mm Mg EDTA, 0.2 mm 3HTdR (5 Ci/mmol) or 0.1 mm 3H-araC (35 Ci/mmol), respectively, were added to 100 ␮l cell extract and incubated at 37°C for 1 h. The reaction was stopped by incubation of the samples at 100°C for 2 min. After precipitation of the proteins by centrifugation for 3 min at 2500 g and 4°C, 20 ␮l of the clarified reaction mixture was spotted on DE81 anion exchange filter discs (Whatman), and washed twice with 0.1 mm ammoniumformiat (5 ml) and H2O (5 ml), and once with 96% ethanol. Radioactivity of the phosphorylated nucleosides was counted in a LKB fluid scintillation counter. All experiments were performed in triplicate.

Deoxycytidine deaminase assay For DCD assay, 90␮l of reaction buffer (final concentrations: 1.9 mm Tris HCl pH 8.0, 60 nm deoxycytidine) and deoxy (53 H) cytidine (0.006 ␮Ci/ml, 22 Ci/mmol, Amersham Buchler) were added to 10 ␮l cell extract and incubated at 37°C for 60 min. The reaction was terminated by addition of 50 ␮l 1.2 m trichloracetic acid and the mixture was given on a 0.5 × 7 cm column of Dowex resin (AG 50W-XA, BioRad, Richmond, USA), which was subsequently washed with distilled water (2 volumes). The resulting product 3H-deoxyuridine was eluted with the void volume and radioactivity was determined in a liquid scintillation counter. All experiments were performed in triplicate.

Statistical methods Comparisons between two groups of patients were made by the Wilcoxon-test, P values ⬍0.05 were considered significant. Survival differences were estimated using the Kaplan– Meier method. Data were analyzed using the PC-Statistik program (Lizenzagentur Lambda, Graz and TopSoft, Version 2.05, Hannover, Germany). Leukemia


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Table 1

Patient characteristics

Number of patients Age (years) Sex: Bone marrow blast infiltration (%) FAB subtype

Table 2 Relation of karyotype analyses with proliferative activity of AML blasts 91 range: 19–80 median: 51 age ⬎60: 27 male: 49 female: 42 range: 70–100 median: 90 MO: 2 M1: 12 M2: 20 M3: 1 M4: 36 M5: 18 M6: 2

Results Pretherapeutic parameters of araC metabolism and proliferative activity were obtained from 94 patients with newly diagnosed AML. Cytogenetic analyses were successfully performed in 91 of these cases. Patient characteristics are depicted in Table 1.

Karyotype and clinical outcome The 91 cases in whom karyotypes were obtained were grouped into the previously defined prognostic subgroups: (I) normal karyotype (n = 38); (II) favorable karyotypes (n = 16); (III) unfavorable karyotypes (n = 20); (IV) karyotypes of uncertain prognostic significance (n = 17). Patients with a favorable karyotype had a higher CR rate (92%) and experienced a longer median survival as compared to patients of any other cytogenetic subgroup (Figure 1). CR rates were 63% for group I, 71% for group III and 77% for group IV, median survival times were 13.5 months for group I, 27 months for group II, 5 months for group III and 18.5 months for group IV, respectively.

Figure 1 Leukemia

Cytogenetic subgroup

I II III IV a

n

34 13 16 11

3

H-TdR (pmol/105 cells)

Median

Range

0.7 2.48 0.51 0.98

0.0–6.7 0.28–25.8a 0.04–7.62 0.0–4.0

P = 0.0096 (group II vs group III/Wilcoxon-test).

Karyotype and proliferative activity Karyotype analyses were related to the proliferative activity of AML blasts and showed a significantly higher incorporation of 3H-TdR into blast cells from patients with favorable karyotypes as compared to specimens from cases with unfavorable cytogenetics (P = 0.0096) (Table 2).

Karyotype and parameters of araC metabolism The determined pretherapeutic parameters of proliferative activity and araC metabolism are summarized in Table 3 and Table 3 Pretherapeutic parameters of proliferative activity and araC metabolism

Parameter

n

(i) 3H-TdR (pmol/105 cells) ara-C incorporation (pmol/105 cells) (ii) Poly ␣ (pmol/min/mg protein) TK (pmol/min/mg protein) DCK (pmol/min/mg protein) DCD (nmol/min/mg protein)

74 39

Relationship between karyotype analyses and clinical outcome of patients.

44 50 52 54

Median

Range

0.7 0.0–25.8 0.059 0.001–0.449 1.58 5.6 59.4 0.75

0.0–8.9 0.5–33.8 4.1–199.8 0.02–29.5


Table 4 Relation of karyotype analyses with parameters of araC metabolism of AML blasts

Parameter

AraC incorporation (pmol/105 cells) Poly ␣ (pmol/min/mg prot) DCK (pmol/min/mg prot) DCD (nmol/min/mg prot)

Cytogenetic subgroup

n

Median

Range

I II III IV I II III IV I II III IV I II III IV

18 10 5 6 20 7 7 10 24 8 9 11 25 9 9 11

0.057 0.048 0.09 0.043 1.18 2.37 1.8 1.37 60.8 63.2 44.8 64.6 0.70 1.64 0.81 0.48

0.001–0.22 0.015–0.11 0.024–0.45 0.008–0.10 0.25–5.34 1.2–8.9 0.12–6.7 0.0–5.3 4.65–199.8 4.5–116.3 4.1–120.3 6.9–168.1 0.02–8.45 0.029–5.57 0.046–29.5 0.02–8.61

whereas the proportion of responses to GM-CSF was only 23% in group II. Sensitivity to GM-CSF in group II and group III was obviously related to the proliferative activity of AML blasts. Taking the overall median increase in 3H-TdR incorporation of 1.7fold as arbitrary cut-point for high or low blast cell proliferation a pattern of response could be identified. Hence, 54% of patients with favorable karyotypes have a higher than median proliferative activity and do not respond to GM-CSF stimulation whereas 56% of cases with unfavorable cytogenetics display an inverse relation with a lower than median proliferative activity and a significant response to GM-CSF (Table 6). From these data, a certain association between karyotype, proliferative activity and stimulation by GM-CSF emerges. Hence, a favorable karyotype is characterized by a high proliferative activity and a low response to GM-CSF stimulation. Inversely, unfavorable cytogenetics are associated with a low proliferative activity but a high sensitivity to GM-CSF stimulation.

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Discussion indicate a broad interpatient variation. Table 4 indicates that no significant differences were observed among the cytogenetic subgroups for 3H-araC incorporation into DNA. Similarly, the activities of poly ␣, DCK and DCD were evenly distributed.

Karyotype and response to GM-CSF Blast cells of 44 from 74 analyzed patients (59%) were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation ⬎1.5-fold over control values after the 48 h GM-CSF exposure. Response to GM-CSF differed between individual cases with changes of 3H-TdR incorporation ranging from 0.0- to 26.7-fold over baseline values (median 1.7-fold). Patients with a favorable karyotype (group II) had a significantly lower sensitivity to GM-CSF (median: 1.20-fold, range: 0.82–2.8) as compared to blast cells with normal karyotypes (group I, median: 1.95-fold, range: 0.0–26.7) (P = 0.04) or unfavorable cytogenetics (group III, median: 2.28-fold, range: 0.87–8.5) (P = 0.013) (Table 5). Correspondingly, the highest percentage of GM-CSF sensitive cases (81%) was detected in group III (unfavorable karyotype), Table 5 blasts

Relation of karyotype and sensitivity to GM-CSF of AML

Karyotype

Normal Favorable Unfavorable Uncertain

n

34 13 16 11

GM-CSF sensitivity Response (%)

Median 3H-TDR increase (fold of control values)

64 23 81 63

1.95a 1.2 2.28b 1.6

Response to GM-CSF was measured by the rate of 3H-TdR incorporation after the 48 h GM-CSF exposure, compared to the rate of 3H-TdR incorporation of control samples. a P = 0.04 (group II vs group I/Wilcoxon-test) b P = 0.013 (group II vs group III/Wilcoxon-test)

This recent study is based on the broadly accepted relevance of cytogenetics as the main determinant for treatment outcome in AML and attempts to elucidate the mechanisms underlying the different biology of cytogenetic subgroups in greater detail. The current data clearly show that cytogenetic subgroups differ significantly in spontaneous proliferative activity. Hence, patients with favorable karyotypes had a significantly higher proliferative activity as compared to cases with unfavorable cytogenetics. This finding may explain the previously reported association between a high spontaneous 3H-TdR incorporation and a good response to chemotherapy.43 On the other hand, an inverse association was found between proliferative activity and the ability of leukemic blasts to increase their proliferation rate in response to hematopietic growth factors such as GM-CSF in particular. The biology that underlies the relation between proliferative activity and response to therapy is still not well understood. In a previous study we have shown, that an increase in 3HTdR incorporation leads to an increased 3H-araC uptake. However, the lacking correlation of 3H-araC incorporation in vitro with treatment response implies that additional mechanisms of araC cytotoxicity exist that are not mediated by araCTP and araC incorporation into DNA. It is well estabTable 6 Association of karyotype with proliferative activity and sensitivity to GM-CSF stimulation

Group I 3 H-TdR ⬍median 5 (15%) GM-CSF response − 13 (38%) 3 H-TdR ⬍median GM-CSF response + 7 (21%) 3 H-TdR ⬎median GM-CSF response − 9 (26%) 3 H-TdR ⬎median GM-CSF response + Total 34

Group II

Group III

Group IV

3 (23%) 1 (8%)

3 (19%) 9 (56%)

1 (9%) 2 (18%)

7 (54%)

1 (6%)

3 (27%)

2 (15%)

3 (19%)

5 (45%)

13

16

11

Blast cells were cultured for 48 h in the presence or absence of GM-CSF and 3H-TdR was added for an additional 4 h. Sensitivity to GM-CSF was defined by an increase in 3H-TdR incorporation ⬎1.5-fold over control values after the 48 h GM-CSF exposure. Leukemia


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lished, that many chemotherapeutic agents kill tumor cells by initiating apotosis. On the other hand, there are a variety of anti-apoptotic signals such as bcl2-expression,62 bcr-abl,63 loss of p5364 that have been associated with drug resistance in AML. However, these distinct events are relatively uncommon in newly diagnosed de novo AML. Therefore, the intrinsic susceptibility of AML cells to apoptosis is probably a function of interactions among multiple signals that influence apoptosis.65 It is tempting to speculate that sensitivity of araC is increased by growth factors as a result of conflicting signals that simultaneously promote growth arrest and cell cycle progression.66,67 These unresolved problems emphasize the need to apply multifactorial analyses for the characterization of leukemic cells and to determine the interrelation and hierarchy among individual parameters. Even the grouping of cytogenetic abnormalities according to treatment outcome must be considered as merely descriptive and still insufficient to direct treatment strategies. This fact is supported by recent reports about a different response of cytogenetic subgroups to highdose araC. Hence, among cases with so-called favorable cytogenetics patients with inv 16 experienced an improved remission duration after consolidation with high dose araC while no benefit was observed in cases with t(8;21). In contrast, the application of high-dose araC during induction therapy resulted in a more favorable prognosis of patients with t(8;21), while cases with inv16 showed no benefit from such treatment.15,68 These data indicate that cytogenetics alone are still insufficient for the selection of therapy and that parameters of araC metabolism may be additional and possibly independent determinants. Differences among cytogenetic subgroups have also been observed for their response to hematopoietic growth factors. Hence, Kita et al69 found that blasts with t(8;21) reveal a poor response to GM-CSF due to a low number of GM-CSF receptors. Cremer et al70 recently demonstrated a preferential stimulation of cells with trisomy 8 as compared to normal hematopoietic progenitors after exposure to GM-CSF. In the current study cases with favorable karyotypes had a significantly lower response to GM-CSF as compared to patients with unfavorable cytogenetics which was associated with an inverse relation to proliferative activities. These results confirm the previous observation that the sensitivity of leukemic blasts to hemopoietic growth factors is associated with a low proliferation rate which was inversely related to early response to induction therapy.43 Based on these data it is tempting to speculate that patients with unfavorable cytogenetics may be preferential candidates for priming concepts while the high spontaneous proliferation of blast cells with favorable cytogenetics prohibits a further increase by GM-CSF stimulation. These questions need to be addressed in subsequent studies which might finally result in pathogenesis and risk-orientated treatment strategies.

Acknowledgements

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This study was supported by a grant from the Dr MilderedScheel Stiftung fu¨r Krebsforschung, Germany (W 131/94/Hi7).

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