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The Journal of Immunology, 2006, 176: 1759-1768.
Copyright © 2006 by The American Association of Immunologists

Immunological Characterization and Antibacterial Function of Persisting Granulocytes in Leukemic Patients Receiving Pulse Cytosine Arabinoside-Consolidation Chemotherapy on Days 1, 3, and 51

Maria-T. Krauth*, Stefan Florian*, Alexandra Böhm*, Karoline Sonneck*, Hermine Agis*, Puchit Samorapoompichit{dagger}, Alexander W. Hauswirth*, Wolfgang R. Sperr* and Peter Valent2,*

* Department of Internal Medicine I, Division of Hematology and Hemostaseology, Center of Excellence in Clinical and Experimental Oncology, Vienna, Austria; and {dagger} Institute of Histology and Embryology, Medical University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
High-dose cytosine arabinoside (HiDAC) and intermediate-dose cytosine arabinoside (IDAC) have been introduced as effective and safe consolidation chemotherapy in acute myeloid leukemia, with relatively low rates of life-threatening infections despite the high total dose of the cytostatic drug. To explore the biological background of low toxicity, we examined the numbers, immunophenotype, and functional properties of granulocytes in patients with acute myeloid leukemia receiving HiDAC or IDAC. Interestingly, the absolute numbers of neutrophils remained >500/µl until day 10 in 92 of 125 (74%) HiDAC cycles and in 106 of 113 (94%) IDAC cycles. As assessed by electron microscopy, these day-10 granulocytes surviving chemotherapy were found to be mature cells containing secondary granules and phagolysosomes. They also expressed opsonization- and phagocytosis-linked surface Ags (C3biR, CR1, C1qR, C5aR, Fc{gamma}RI, Fc{gamma}RII, Fc{gamma}RIII, and G-CSF and GM-CSF receptors) like neutrophils in healthy controls. Moreover, these day-10 neutrophils exhibited oxidative burst activity and took up and digested bacteria in the same way as neutrophils in healthy controls. There was a negative correlation between absolute neutrophil counts and severe infections in HiDAC- and IDAC-treated patients with a later onset of infections in IDAC patients (median: IDAC, day 18; HiDAC, day 16). Together, functionally mature neutrophils are detectable at least until day 10 in patients treated with HiDAC or IDAC, and may explain the relatively low hematologic toxicity of these consolidation protocols. IDAC is a superior protocol in this regard and may therefore be most suitable for elderly patients and those at high risk for severe infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acute myeloid leukemia (AML)3 is characterized by uncontrolled clonal proliferation of myeloid progenitor cells without significant differentiation or maturation (1, 2, 3). The prognosis and clinical picture in AML vary depending on age, deregulated genes, and the specific biological properties of the clone (1, 2, 3, 4, 5). The first important goal in the treatment of AML is to achieve complete remission (CR) by applying induction polychemotherapy. Patients who do not enter CR in response to induction chemotherapy have a grave prognosis (6, 7, 8).

For patients entering CR, it is generally appreciated that postremission therapy with repetitive cycles of conventional chemotherapy (or stem cell transplantation) is required to maintain long-term disease-free survival (DFS) (7, 8, 9, 10, 11). Because AML patients in CR have an uncertain prognosis in most cases but can potentially be cured, consolidation therapy has to meet two important objectives. First, these protocols should contain as much therapy as required to maintain a disease-free state. Second, these protocols should be associated with a low risk of therapy-related mortality. A most important aspect in this regard is life-threatening infections that can occur after chemotherapy during the time of complete aplasia (6, 7, 8, 9, 10). In fact, the time of absolute neutropenia (absolute neutrophil count (ANC) < 500/µl) is known to correlate with the rate of severe, life-threatening infections after chemotherapy (6, 7, 8, 9, 10, 11, 12).

A number of previous and more recent data suggest that 1-beta-D-arabinofuranosyl-cytosine (cytosine arabinoside; ARA-C), a DNA replication inhibitor, is a highly effective antileukemic agent that is useful in the treatment of AML in both the induction and consolidation phase of therapy. In 1994, the Cancer and Leukemia Group B (CALGB) study group introduced the high-dose intermittent (pulse) ARA-C regimen (HiDAC; ARA-C 2 x 3 g/m2 on days 1, 3, and 5) as an effective consolidation treatment for patients with AML aged <60 years (13). However, in patients over the age of 60, severe neurotoxicity was reported (13). Therefore, we have recently dose-modified this protocol for elderly patients with AML by using pulse-ARA-C at 2 x 1 g/m2 on days 1, 3, and 5 (intermediate-dose cytosine arabinoside; IDAC) (14). Both regimens were found to be effective and well-tolerated consolidation therapies, with a relatively low rate of severe infections and low mortality (13, 14, 15). However, the factors that contribute to the relatively low toxicity and low mortality in these protocols remain so far unknown.

In this study, we show that in patients treated with IDAC or HiDAC, the ANC often remain >500/µl for a prolonged time period after chemotherapy, i.e., until day 10 or even day 14. We also show that these persisting granulocytes are functionally and phenotypically mature cells and can kill bacteria effectively, and thus may contribute to host defense after chemotherapy and thereby to the relatively low rate of life-threatening infections in these patients. Finally, our data show that IDAC patients have higher numbers of ANC compared with HiDAC patients on day 10 and day 14, suggesting that IDAC may be a most suitable protocol for older patients and those who have a high risk for severe infections.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients’ characteristics

A total number of 199 patients with de novo AML (95 patients aged <60 years, median age 42, f/m (female/male) ratio 1.4:1; 104 patients aged ≥60 years, median age 71, f/m ratio 0.7:1) were examined. Of these, 125 patients (<60 years of age, n = 66; ≥60 years of age, n = 59) achieved hematologic CR after standard induction therapy described in detail elsewhere (14). These 125 CR patients received consolidation and were analyzed in follow-up examinations between October 1994 and April 2004 in a single center (Vienna University Hospital, Vienna, Austria). Patients with a history of a preceding hematologic neoplasm, radiation, or chemotherapy (secondary AML) were excluded. Diagnoses were established according to criteria provided by the French-American-British (FAB) cooperative study group (16, 17, 18). Responses to chemotherapy were determined according to published guidelines (19, 20). The patients’ characteristics are shown in Table I.


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  		Table I. Characteristics of AML patients receiving chemotherapya

 
Consolidation regimens: HiDAC and IDAC

In all patients, written informed consent was obtained before chemotherapy. The consolidation regimen was identical with the protocol described by the CALGB study group (13), with an age adaptation for elderly patients (≥60 years) as reported previously (14). In particular, patients aged <60 years received up to four cycles of HiDAC (2 x 3 g/m2/day ARA-C i.v. as 3-h infusions in 12 h-intervals, on days 1, 3, and 5) (13, 15). In the elderly patients (≥60 years old), consolidation consisted of up to four cycles of IDAC: 2 x 1 g/m2/day ARA-C i.v. on days 1, 3, and 5 (3-h infusions in 12-h intervals) (14). Red cell concentrates were given to maintain hemoglobin levels >8.0 g/dl. Platelets were transfused to keep the platelet count >10,000/µl (in case of severe infection or mucositis, platelets were kept >20,000/µl). In addition, patients received prophylactic trimethoprim and ophthalmic steroid drops during therapy. In case of a severe infection during one of the preceding chemotherapy cycles, they also received prophylactic antimycotics. Recombinant human G-CSF was not administered routinely. However, G-CSF (Neupogen; 30 million units per day, s.c.) was administered in case of suspected or established severe infection during prolonged neutropenia or a known history of a severe infection occurring during one of the preceding chemotherapy cycles. Overall, G-CSF was applied in 76 of 199 HiDAC cycles (38%) and in 80 of 184 (43%) IDAC cycles. If applied, the cytokine was always initiated after day 10.

Determination of ANC during HiDAC and IDAC, and evaluation of toxicity

In each chemotherapy cycle (HiDAC or IDAC), ANC were serially determined until hematologic recovery. Hematologic toxicity was examined by analyzing the duration of neutropenia (ANC <500 cells/µl) and days of neutropenic fever. In addition, the onset (first day) of occurrence of neutropenic fever was documented.

Examination of granulocytes on day 10 after start of chemotherapy

To determine phenotypic and functional properties of peripheral blood granulocytes after chemotherapy, blood was drawn on day 10 after start of consolidation therapy in eight patients receiving HiDAC and eight patients receiving IDAC (Table II). In addition, granulocytes obtained from five healthy volunteers were examined. Granulocytes were enriched by dextran sedimentation and examined morphologically by light microscopy on Wright-Giemsa-stained cytospin preparations. In four patients, the ultrastructural features of isolated neutrophils were examined by electron microscopy (see below).


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  		Table II. Characteristics of patients in whom granulocyte function was examined

 
Surface marker studies

Expression of host defense-related molecules involved in opsonization or/and phagocytosis on day-10 granulocytes was examined by mAbs and flow cytometry. All of the Abs were purchased from companies listed in Table III. Granulocytes were analyzed in heparinized whole blood samples obtained from five healthy controls and 16 patients receiving chemotherapy (HiDAC, n = 7; IDAC, n = 9) on day 10. Cell staining and flow cytometry were performed essentially as described previously (21). In brief, whole blood samples containing 1 x 106 leukocytes were incubated with mAbs (1 µg) at 4°C for 30 min. Then, cells were washed in PBS. In case of unconjugated mAbs, cells were incubated with a second step FITC-labeled goat anti-mouse Ab at 4°C for 30 min and washed. After Ab staining, cells were incubated in erythrocyte lysis buffer (BD Biosciences) at 4°C for 30 min. Ab reactivity was controlled by isotype-matched control Abs. Ab reactivities on leukocyte surface membranes were quantified on a FACScan (BD Biosciences). A specification of mAbs applied is given in Table III.


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  		Table III. Specification of mAbs

 
Examination of granulocytes by the NBT reduction test

Dextran-enriched day-10 granulocytes were examined for burst activity by the NBT reduction assay essentially as described previously (22). In brief, enriched cells were resuspended in RPMI 1640 medium with 10% FCS and incubated with an equal volume of PBS containing 0.2% NBT (Sigma Chemical) and 2 µg/ml freshly diluted 12-tetra-decanoylphorbol-13-acetate at 37°C for 20 min. After incubation, cytospin preparations were prepared and viewed under a light microscope.

Evaluation of bacteria uptake in neutrophils by flow cytometry

To demonstrate that day-10 neutrophils are capable of contributing to host defense, we analyzed the uptake of FITC-labeled Escherichia coli bacteria (Molecular Probes) in these cells by flow cytometry. Granulocytes were analyzed in 10 patients receiving HiDAC or IDAC as well as in 10 healthy controls. Whole blood (70–300 µl; adjusted to a total leukocyte number of 0.5–1.0 x 106/sample) was incubated with bacteria (25 bacteria per white blood cell) at either 37°C or 4°C (control) for 10 min. Thereafter, blood cells were washed in PBS at 4°C and then incubated for 30 min with a PE-labeled mAb against CD114 and a FITC-labeled CD14 mAb for detection of neutrophils and monocytes, respectively. After staining, cells were fixed in erythrocyte lysis buffer at 4°C for 30 min, washed in PBS, and then exposed to propidium iodide. Two-color flow cytometry was performed on a FACScan (BD Biosciences). Specific uptake of E. coli bacteria was calculated from differences in mean fluorescence intensities (MFI) found between cells exposed to bacteria at 4°C (nonspecific uptake/binding of bacteria) and those exposed to bacteria at 37°C.

Evaluation of bacteria uptake and killing in neutrophils by electron microscopy

Bacteria uptake and killing was examined in day-10 neutrophils (one patient receiving HiDAC and one receiving IDAC) and normal neutrophils (one healthy donor) by electron microscopy. For this purpose, dextran-isolated neutrophils (1 x 107 in each sample) were incubated with E. coli bacteria (25 bacteria per leukocyte) in autologous serum for 0, 10, 30, and 180 min at 37°C. After incubation, cells were recovered and fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.025% CaCl2, buffered in 0.1 mol/l sodium cacodylate buffer (pH 7.4) at room temperature for 60 min. Then, cells were washed three times in 0.1 mol/l sodium cacodylate buffer, suspended in 2% agar, and centrifuged. The pellets were postfixed with 1.3% OsO4 (buffered in 0.66 mol/l collidine) and stained en bloc in 2% uranyl acetate and sodium maleate buffer (pH 4.4) for 2 h at room temperature. Then, pellets were rinsed, dehydrated in alcohol series, and embedded in EPON 812. Ultrathin sections (85 nM) were cut and placed on gold grids. Sections were contrasted in uranyl acetate and lead citrate and viewed in a JEOL 1200 EX II transmission electron microscope (JEOL). Opsonized and ingested E. coli bacteria were identified by their typical morphology and size.

Statistical analysis

Patients had to have completed the last cycle of their consolidation chemotherapy to be included in statistical analyses. The product limit method of Kaplan-Meier was applied to analyze the probability of survival and DFS. Differences were considered to be significant when the p value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Numbers of chemotherapy cycles administered

Independent of age and type of consolidation (HiDAC, n = 66 vs IDAC, n = 59), all patients (n = 125) were scheduled to receive four cycles of consolidation-chemotherapy. All four courses were administered in 63 of the 125 patients (50.4%), i.e., in 34 of 66 patients below 60 years of age (51.5%), and in 29 of 59 patients over 60 years of age who received IDAC (49.2%). Twenty-seven of the 125 patients (21.6%) received three cycles (12 of 66 HiDAC patients (18.2%), and 15 of 59 IDAC patients (25.4%)), and 15 of 125 patients (12.0%) received two cycles (7 of 66 HiDAC patients (10.6%), and 8 of 59 IDAC patients (13.6%)). In 20 of 125 patients (16.0%), the treatment had to be discontinued after the first cycle (13 HiDAC patients and seven IDAC patients). Severe infection was the major cause to discontinue consolidation therapy.

Numbers of neutrophil granulocytes in patients receiving HiDAC or IDAC

After initiation of consolidation chemotherapy (HiDAC or IDAC), leukocyte counts and ANC decreased during the first 2 wk. The ANC nadir was reached in the third week after start of chemotherapy in almost all patients (Fig. 1A). A most remarkable phenomenon was that leukocytes and ANC decreased slowly and often showed unexpected short-lived spontaneous increases between days 3 and 14, resulting in relatively high neutrophil counts until day 10 (and often until day 14) in these patients (Fig. 2). In a majority of the HiDAC cycles applied (74%), ANC on day 10 exceeded 500/µl, whereas on day 14, ANC remained >500/µl in only 12% of all chemotherapy cycles applied (Fig. 1B). In the IDAC group, however, ANC remained >500/µl in the vast majority of the cycles on day 10 (94%), and in as many as 48% of the cycles, ANC were >500/µl on day 14 (Fig. 1B). Examples for the differences in ANC on day 14 in (individual) patients receiving HiDAC (Fig. 2, A and B) as opposed to those receiving IDAC (Fig. 2, C and D) is shown in Fig. 2. The median number of days with absolute neutropenia (ANC < 500) amounted to 12 in patients receiving HiDAC, and to 9 days in those receiving IDAC.


Figure 1
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  		FIGURE 1. Numbers of neutrophils during consolidation. ANCs were determined serially before, during, and after consolidation therapy using ARA-C at days 1, 3, and 5. The protocol was age-adapted: AML patients <60 years of age received HiDAC (•-•), and patients aged ≥60 years received IDAC ({blacktriangleup}-{blacktriangleup}). A, Median ANC during and after therapy in all patients and cycles ({blacksquare}-{blacksquare}) as well as in the subgroups analyzed (HiDAC, all cycles; and IDAC, all cycles). B, The percentage of cycles in which patients exhibited ANC > 500 for each day after start of chemotherapy. Again, all patients ({blacksquare}-{blacksquare}), those receiving HiDAC (•-•) and those receiving IDAC ({blacktriangleup}-{blacktriangleup}), are shown. As visible, ANC remained at a relatively high level until day 14 after start of IDAC, i.e., in nearly 50% of all cycles applied.

 

Figure 2
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  		FIGURE 2. Neutrophil counts in individual patients receiving HiDAC or IDAC. ANCs were calculated from white blood cell counts (WBC) and differential counts. WBC and ANC were serially determined during and after chemotherapy in patients receiving HiDAC (A and B) or IDAC (C and D). As visible, neutrophil counts often increased between days 5 and 10 after the start of HiDAC or IDAC. This unexpected increase in neutrophils was found to be a recurrent phenomenon seen in most patients. A remarkable observation was that in patients receiving IDAC (C and D), but not in those receiving HiDAC (A and B), ANC usually remained >500/µl on day 14.

 
Morphology and ultrastructure of neutrophils on day 10 after start of chemotherapy

An important next question to be answered was whether day-10 granulocytes represent morphologically and functionally mature neutrophils. As assessed by light microscopy (Wright-Giemsa stains) and electron microscopy, granulocytic cells in patients receiving HiDAC or IDAC appeared to be mature cells with segmented nuclei, pale cytoplasm, and neutrophilic secondary granules (Fig. 3). By electron microscopy, these cells contained secondary granules and phagolysosomes similar to neutrophils in healthy controls (Fig. 3). No morphologic or ultrastructural differences were found when comparing day-10 granulocytes in patients receiving HiDAC with those in patients receiving IDAC. Thus, granulocytes in patients after HiDAC or IDAC on day 10 after start of chemotherapy resemble morphologically mature neutrophils.


Figure 3
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  		FIGURE 3. Morphology and ultrastructure of day-10 neutrophils in a patient receiving IDAC. A, The morphology of day-10 granulocytes in a patient with AML receiving IDAC for consolidation (Wright-Giemsa staining). B, Electron microscopic features of a neutrophil granulocyte in an AML patient in CR receiving IDAC. Granulocytes were obtained on day 10 after start of chemotherapy. The granulocyte was found to be mature and to contain a segmented nucleus as well as secondary neutrophil granules.

 
Expression of host defense-related cell surface Ags on day-10 neutrophils

To determine whether day-10 granulocytes in patients receiving HiDAC or IDAC display host defense-related surface Ags, flow cytometry analyses were performed using mAbs against various complement, Fc{gamma}, and cytokine receptors. In these experiments, day-10 neutrophils were found to express Lewisx (CD15), the C3biR (CD11b), CR1 (CD35), C5aR (CD88), C1qR (CD93), Fc{gamma}RI (CD64), Fc{gamma}RII (CD32), Fc{gamma}RIII (CD16), the GM-CSF receptor {alpha}-chain (CD116), and the G-CSF receptor (CD114) (Fig. 4). No differences in expression of host defense-related cell surface Ags were found when comparing day-10 neutrophils in patients receiving HiDAC or IDAC with neutrophils obtained from healthy controls (Fig. 4). In addition, no differences in expression of host-defense Ags were seen when comparing day-10 neutrophils of patients receiving HiDAC with day-10 neutrophils in patients receiving IDAC (data not shown). These data suggest that day-10 granulocytes in patients receiving IDAC or HiDAC are phenotypically mature cells expressing all surface Ags required for bacteria recognition, opsonization, and bacteria uptake.


Figure 4
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  		FIGURE 4. Immunophenotype of day-10 neutrophils in patients receiving HiDAC or IDAC. Expression of host defense-related cell surface Ags on day-10 neutrophils in patients receiving pulse ARA-C on days 1, 3, and 5 (HiDAC, n = 7; IDAC, n = 9) ({blacksquare}) as well as on neutrophils obtained from healthy volunteers (n = 5) ({square}). Expression of cell surface Ags was analyzed by mAbs and flow cytometry. Results represent MFIs obtained in each group and are expressed as mean ± SD of results from five (healthy controls) or 16 (patients) samples.

 
Evaluation of bacteria uptake and bacteria killing in day-10 neutrophils

To confirm that day-10 neutrophils in patients receiving HiDAC or IDAC are indeed capable of contributing to host defense, we analyzed bacteria uptake and bacteria killing in these cells. As assessed by flow cytometry, we were able to show that isolated day-10 neutrophils in both groups of patients are capable of taking up fluorescence-labeled E. coli bacteria (Fig. 5). The quantitative uptake of bacteria in these cells (determined by MFI) was slightly higher compared with bacteria uptake in neutrophils obtained from healthy controls, although the difference did not reach statistical significance (p > 0.05) (Fig. 5E). To further demonstrate bacteria uptake and bacteria degradation in neutrophils, electron microscopy was applied. In these analyses, a time-dependent uptake and degradation of bacteria in neutrophils could be demonstrated (Fig. 5, F–I). Within 10 min, most bacteria were found in phagolysosomes of day-10 neutrophils. After 180 min, most bacteria had been digested (Fig. 5, F–I). There were no differences in time kinetics or quality of uptake of bacteria when neutrophils of patients with HiDAC were compared with those obtained from patients receiving IDAC or neutrophils in healthy controls. Also, there were no subsets of neutrophils in which bacteria uptake or degradation was slow or incomplete in the patients examined. In line with this observation, we were also able to show that day-10 neutrophils exhibit oxidative burst activity as determined by the NBT test. Notably, almost all granulocytic cells obtained on day 10 from patients receiving either HiDAC or IDAC (as well as neutrophils from controls) were found to stain positive for NBT (Fig. 6). All in all, these results show that neutrophil granulocytes on day 10 in AML patients receiving HiDAC or IDAC are able to take up and to digest E. coli bacteria, and thus are immunocompetent cells likely to participate in host defense in vivo.


Figure 5
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  		FIGURE 5. Uptake and digestion of bacteria by day-10 neutrophils. A–D, Flow-cytometric measurement of bacteria uptake by day-10 neutrophils in a patient receiving HiDAC (A and B) using FITC-conjugated E. coli bacteria. Leukocytes from a healthy donor are also shown (C and D). Cells were incubated with FITC-labeled E. coli bacteria at either 37°C (B and D) or 4°C (A and C). Based on their light scattering properties, neutrophils were easily identified and were found to take up bacteria. E, Comparison of bacterial uptake by neutrophils in 10 normal donors and 10 AML patients (day-10 neutrophils). Bacteria uptake is expressed as MFI ratio, MFI of bacteria-laden neutrophils (kept at 37°C):MFI of neutrophils kept with bacteria at 4°C (nonspecific bacteria uptake). Results represent the mean ± SD of MFI ratios obtained in the 10 donors in each group. F–I, Electron microscopic examination of bacteria uptake and bacteria digestion in day-10 neutrophils. Neutrophils obtained from an AML patient on day 10 after start of chemotherapy (HiDAC) were incubated with E. coli bacteria for 10 min (F), 30 min (G), or 180 min (H and I). After incubation, cells were examined serially by electron microscopy. As visible, the E. coli bacteria were ingested and incorporated into phagolysosomes in day-10 neutrophils within 30 min. After 180 min, most bacteria were found to be digested within these cells.

 

Figure 6
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  		FIGURE 6. Functional properties of day-10 neutrophils: evaluation of oxidative burst activity. Day-10 neutrophils were obtained from a patient receiving HiDAC and stained with Wright-Giemsa (A) and subjected to the NBT reduction test (B), an indicator assay for the oxidative burst known to correlate with antibacterial activity. As visible, neutrophils on day 10 were enzymatically active cells. In fact, almost all of the enriched neutrophils stained positive for NBT.

 
Clinical significance of persistence of functionally active neutrophils in patients receiving HiDAC or IDAC

To demonstrate the clinical significance of functionally active, bacteria-killing neutrophils until day 10 (and even on day 14 in many patients receiving IDAC) after start of consolidation chemotherapy, we determined the time (days) of absolute neutropenia, time of severe infections (days with fever >38°C), and the first day of occurrence of infection-associated fever in patients receiving HiDAC or IDAC.

A total of 190 (IDAC, n = 88 and HiDAC, n = 102) of 383 consolidation cycles (49.6%) were accompanied by an infection with fever (>38°C). A summary of neutropenic infections is shown in Table IV. As mentioned above, the number of days with severe neutropenia (ANC < 500/µl) was lower in patients receiving IDAC (9 days) compared with those with HiDAC (12 days). The time period (median days) of febrile neutropenia was also shorter in patients treated with IDAC (2.2 days) compared with patients receiving HiDAC (3.6 days). Because of the different age of the patients in the two treatment arms, however, no statistical analysis was performed with these data.


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  		Table IV. Neutropenic infections occurring in patients receiving HiDAC or IDAC

 
When analyzing the time point at which fever associated with infection was first noted after the start of chemotherapy, it was found that the onset of febrile neutropenia was later in patients with IDAC (day 18) than in those receiving HiDAC (day 16). There was no significant difference in neutrophil recovery after chemotherapy-induced aplasia when comparing the two groups of patients (Fig. 1A).

Together, these results suggest that higher doses of ARA-C are associated with an earlier onset of severe neutropenia, longer time of neutropenia, and higher rate of severe infections compared with IDAC.

Survival in patients receiving HiDAC or IDAC

The overall survival in all CR patients is shown in Fig. 7A. The median overall survival in patients who achieved a CR and were treated with IDAC was found to be 24.9 mo, and the probability to be alive at 5 years was 30%. CR patients receiving HiDAC had a median overall survival of 27.4 mo and a probability of 45% to be alive after 5 years (Fig. 7A). The estimated probability to remain free from recurrent leukemia and to stay alive after entering CR (DFS) was 38% for patients receiving HiDAC and 21.1% for those receiving IDAC at 5 years (Fig. 7B). The median DFS was 12.2 mo for HiDAC patients and 14.7 mo for patients receiving IDAC. The median continuous CR (CCR) rate was 14.6 in patients receiving HiDAC and 15.9 in those treated with IDAC, with a probability of CCR of 42.3% (HiDAC) and 27.6 (IDAC) at 5 years, respectively (Fig. 7C).


Figure 7
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  		FIGURE 7. Survival of patients receiving HiDAC and IDAC. The Kaplan-Meier method was applied to determine overall survival (A), DFS (B), and CCR (C). For the evaluation of DFS and CCR, the patients who received a stem cell transplant were censored.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
ARA-C is a highly effective antileukemic agent that has successfully been used in the treatment of AML for several decades. Repetitive cycles of HiDAC (ARA-C, 3 g/m2 twice a day) on days 1, 3, and 5 were introduced as an effective consolidation for patients with AML by the CALGB study group in 1994 (13). To prevent chemotherapy-induced neurotoxicity, we have recently dose-modified this protocol for elderly patients using pulse ARA-C at 1 g/m2 twice a day on days 1, 3, and 5 (IDAC) (14). Both chemotherapy regimens were found to be highly effective and to exhibit a relatively low rate of severe infections and low rate of chemotherapy-associated mortality (13, 14, 15). In this study, we attempted to define factors and mechanisms that underlie the relatively low rate of life-threatening infections seen in these patients. The results of our study show that neutrophils in these patients often remain above 500/µl until day 10 or even day 14, and that these neutrophils exhibit potent antibacterial activity. IDAC appears to be superior to HiDAC in this regard and, therefore, may be judged as ideal consolidation for elderly patients and those at high risk for severe infections.

Because AML patients in CR usually have an uncertain prognosis but can potentially be cured (6, 7, 8, 9, 10, 11), consolidation therapy has to meet two important objectives: first, therapy should maintain a disease-free state; and second, these regimens should be associated with a low risk of therapy-related mortality. A most important aspect in this regard is life-threatening infections that can occur during the time of chemotherapy-induced aplasia (6, 7, 8, 9, 10). In fact, the time of absolute neutropenia (ANC < 500/µl) is known to correlate with the rate of severe (life-threatening) infections after chemotherapy (6, 7, 8, 9, 10, 11, 12). Our data are in line with this notion and show that the time of complete aplasia correlates with occurrence of severe infections in patients treated with HiDAC or IDAC. In contrast, however, both protocols also seem to be effective with regard to long-term, AML-free survival (13, 14, 15). This is of particular importance for the IDAC protocol, because this protocol contains significantly lower amounts of ARA-C compared with the HiDAC regimen (14). When considering long-term survival, another important factor may be the number of consolidation cycles that can be applied in CR. In the current study, four consolidation cycles were aimed to be administered, and in the majority of the patients, three or four cycles could indeed be applied. The relatively large group (high percentage) of patients receiving three or four cycles of consolidation chemotherapy may in turn be explained by the relatively low rate of life-threatening infections that occurred in these patients. In fact, severe life-threatening infection was the major cause to withdraw patients from further consolidation. Thus, the low rate of neutropenic infections in patients receiving HiDAC or IDAC seems a most important aspect contributing to the rather good outcome in DFS, CCR, and overall survival in our patients (14, 15). It was therefore of particular importance to ask for mechanisms underlying the low rate of infections in these patients.

A first important clue to this question was the observation that, in both consolidation protocols, the numbers of neutrophils remained above 500/µl until day 10 in a majority of all cycles administered. In the IDAC group, neutrophils even remained >500/µl until day 14 in almost half of all cycles applied. In the HiDAC group, however, ANC remained >500/µl on day 14 in only 12% of all cycles. This divergent outcome is most probably due to the different dosage of ARA-C in the two groups. An influence of G-CSF on this outcome can be excluded. In fact, the number (percentage) of patients receiving G-CSF was almost the same in both groups, and, if applied, G-CSF was started only after a severe infection had developed, i.e., after day 14 in almost all cases (G-CSF was not started before day 10). Another explanation for the differences between ANC on day 14 may be age. However, this possibility seems also unlikely because age has no established effect on neutrophil survival or the time of neutrophil maturation, and because neutrophil counts on day 10 or day 14 did not exhibit a linear relation with age in both treatment groups (data not shown).

A remarkable aspect was that neutrophils during chemotherapy did not decrease in HiDAC- or IDAC-treated patients in a strictly time-dependent manner. Rather, blood neutrophils often increased again between days 5 and 14. This unexpected reincrease in neutrophils after IDAC or HiDAC is a remarkable and recurrent phenomenon that was found in most patients. The mechanism for neutrophil-waving could not be clarified, however, and was not seen in other chemotherapy protocols used for consolidation in AML in our department (our unpublished observations). One explanation for the unexpected neutrophil increase around day 10 would be that the pulse-mode of application (days 1, 3, and 5) of chemotherapy resulted in recruitment of neutrophils (progenitors) into a certain phase of the cell cycle, with consecutive accumulation of blood neutrophils. Alternatively, the "neutrophil re-emerging-phenomenon" was due to recruitment of neutrophils from extravascular sites into the blood. An effect of ARA-C on production of growth-enhancing cytokines in vascular cells or other cells in the tissues with consecutive protection of neutrophils from apoptosis may also have occurred. In this regard, it is noteworthy that neutrophils on day 10 were completely viable and functionally mature cells.

When considering neutrophil maintenance as an important factor in contributing to the low rate of life-threatening infections in patients receiving HiDAC or IDAC, it was of pivotal importance to show that these neutrophils indeed are functionally mature cells and are capable of contributing to host defense. To address this issue, day-10 neutrophils were examined in our patients treated with HiDAC or IDAC. In both groups of patients, day-10 neutrophils were found to be mature granulocytic cells containing secondary neutrophil granules and phagolysosomes. In addition, these cells were found to express opsonization- and phagocytosis-related cell surface Ags in the same way as neutrophils from healthy controls. Most importantly, however, we were able to demonstrate that these day-10 neutrophils can take up and kill bacteria as fast and as effectively in IDAC- and HiDAC-treated patients (on day 10) as granulocytes in healthy controls. In addition, these cells are capable of producing and secreting chemotactic peptides such as IL-8 similar to normal neutrophils (data not shown). All in all, our data suggest that neutrophils on day 10 in patients receiving HiDAC or IDAC are morphologically and functionally mature cells, and thus can be expected to contribute to host defense and the relatively low rate of life-threatening infections in these patients. In line with this assumption, the time of onset of neutropenic fever associated with a severe infection was a rather late event in patients receiving IDAC (day 18) when comparing to the onset of severe infections in patients receiving HiDAC (day 16). Thus, the later onset of severe infections correlates with the later onset of absolute neutropenia (<500/µl) in these patients. The later onset of neutropenia also resulted in a shorter time of absolute neutropenia in patients receiving IDAC (9 days) compared with those receiving HiDAC (12 days).

Induction and consolidation of AML in the elderly is becoming an increasingly important aspect in clinical hematology (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). In fact, the majority of all patients with AML are over 60 years of age at diagnosis, and the percentage of patients with AML >60 years of age is constantly rising. Moreover, because of improved supportive care and general therapeutic facilities, more and more of these patients are nowadays judged candidates for curative therapeutic interventions (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). Because these patients cannot tolerate all chemotherapies and therapeutic maneuvers that otherwise can be offered to younger adults (like bone marrow transplantation or very high doses of ARA-C), it seems of great importance to search for alternative treatment strategies for this group of patients. An important aspect in this regard may be the optimal way of consolidation once CR is reached in AML in the elderly (34, 35, 36, 37, 38, 39, 40). Our current protocol may be a valuable contribution in this regard because it shows both sufficient antileukemic activity and a relatively low rate of life-threatening infections with a low rate of treatment-related mortality.

All in all, IDAC seems to be an effective and safe consolidation regimen for elderly patients with AML. In the current study, persistence of functionally active neutrophils could be identified as a relevant mechanism contributing to the relatively low hematologic toxicity and low rate of life-threatening events in these patients. Based on these data, it seems desirable to propose IDAC as a new option for consolidation of AML in the elderly and to apply these and similar protocols to optimize AML therapy. Forthcoming randomized trials should be helpful in this regard.


   Acknowledgments
 
We thank Hans Semper for skillful technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich Grant P-14031 and the Austrian Federal Ministry for Education, Science and Culture Grant GZ 200.062/2-VI/1/2002. Back

2 Address correspondence and reprint requests to Dr. Peter Valent, Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, A-1090 Vienna, Austria. E-mail address: peter.valent{at}meduniwien.ac.at Back

3 Abbreviations used in this paper: AML, acute myeloid leukemia; CR, complete remission; DFS, disease-free survival; ANC, absolute neutrophil count; ARA-C, cytosine arabinoside; HiDAC, high-dose ARA-C; IDAC, intermediate-dose ARA-C; f/m, female/male; FAB, French-American-British; MFI, mean fluorescence intensity; CCR, continuous CR; WBC, white blood cell count. Back

Received for publication April 28, 2005. Accepted for publication November 21, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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