Granulocyte Colony-Stimulating Factor in Acute Myeloid Leukemia

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1 Granulocyte Colony-Stimulating Factor in Acute Myeloid Leukemia Kensuke Usuki," Seiko Iki," Mitsue Endo," Koichi Kitazume," Keiko Ito," Masahiko Watanabe,b Akio Urabea "Division of Hematology, Kanto Teishin Hospital, Tokyo, Japan; bpharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Tokyo, Japan Key Words. G-CSF G-CSF receptor Receptor Acute myeloid leukemia Leukemia Abstract. The clinical application of recombinant human G-CSF in patients with acute myeloid leukemia (AML) has been controversial because it stimulates the in vitro proliferation of leukemic cells. In order to explore the possibility of predicting in vivo leukemic proliferation after G-CSF administration to AML patients by using in vitro assays, we investigated the leukemic blasts of 30 AML patients, including 14 patients who received G-CSF for severe infection associated with neutropenia following chemotherapy (G-CSF group) and 16 patients who did not (control group). Of the 14 patients in the G-CSF group, 9 showed an increase of leukemic blasts in the peripheral blood during G-CSF administration, while 11 of the 16 control patients developed leukemic resurgence. In the G-CSF group, the frequency of leukemic resurgence among patients whose blasts showed dose-dependent proliferation after addition of G-CSF to cultures was similar to that among patients whose blasts did not. In addition, there were no significant differences between the G-CSF and control groups in r3h]thymidine incorporation by leukemic cells and leukemic colony formation after the addition of G-CSF to cultures. The G-CSF receptor affinity of leukemic blasts was significantly higher in the patients with leukemic resurgence (mean dissociation constant [Kd]: 55 pm in the G-CSF group and 63 pm in the control group) than in those without it (101 pm and 96 pm, respectively), and the number of G-CSF receptors per cell was significantly lower when leukemic resurgence occurred (200 in the G-CSF group and 260 in the control group) than when it did not (3400 and 3030, respectively). Correspondence: Dr. Akio Urabe, Division of Hematology, Kanto Teishin Hospital, Higashi- Gotanda, Shinagawa-ku, Tokyo, Japan 141. Received March 27,1995; provisionally accepted May 17, 1995; accepted for publication July 21, OAlphaMed Press /95/$5.00/0 Immunophenotyping (for CD2, CD7, CDlO, CD13, CD19, CD33, CD34, CD71, HLA-DR, glycophorin A and the G-CSF receptor) revealed no significant differences between blasts from the patients with and without leukemic resurgence in the G-CSF group. Thus, we conclude that the in vivo leukemic resurgence during G-CSF administration after chemotherapy for AML was not correlated with the in vitro responsiveness of leukemic blasts to this cytokine or with blast phenotyping data. Leukemic resurgence is likely to occur in patients whose leukemic blasts have fewer numbers of G-CSF receptors with a high affinity irrespective of whether patients receive G-CSF. Introduction Modem combination chemotherapy for adult acute myeloid leukemia (AML) has significantly increased the achievement of complete remission rate and long-term disease-free survival [ 11. This improvement has been particularly attributable to intensified consolidation therapy, such as high-dose cytosine arabinoside [l, 21. However, the myelotoxicity of such treatment results in a substantial morbidity that is primarily related to infectious complications determined by the degree and duration of granulocytopenia. In recent randomized clinical trials, administration of G-CSF after myelosuppressive chemotherapy for solid tumors, lymphoma, and acute lymphoblastic leukemia or after bone marrow transplantation has achieved the marked acceleration of myeloid recovery, as well as a reduction in the incidence and severity of fever and infectious complications [ In addition, the dose intensity of chemotherapy could be increased by using G-CSF [5]. G-CSF has also been given to AML patients after or in combination with intensive STEM CELLS 1995;13:

2 648 G-CSF in Acute Myeloid Leukemia chemotherapy, thereby reducing the incidence of treatment-related infection due to severe neutropenia [ 12, 131. However, the clinical application of G-CSF has been controversial [ 141 because it stimulates the in vitro proliferation of leukemic blast cells in most AML patients [ In fact, the administration of GM-CSF, another potent neutrophil stimulator, has recently been reported to induce marked leukemic proliferation in some Ah4L patients after chemotherapy [18]. In addition, it was previously reported that leukemic proliferation could be induced by G-CSF or GM-CSF in patients with myelodysplastic syndrome and high levels of blasts in the bone marrow or blood [ 19,201. Accordingly, we explored the possibility of predicting the resurgence of AML during G-CSF administration after chemotherapy by using in vitro assays. This retrospective study showed that the in vivo leukemic resurgence during G-CSF treatment was not correlated with blast phenotyping data, or with the in vitro responsiveness of blasts to this cytokine in cell proliferation assay in liquid culture, [3H]thymidine incorporation assay or leukemic colony formation assay. Irrespective of whether patients received G-CSF, the blasts of patients with leukemic resurgence had significantly fewer G-CSF receptors with a significantly higher affinity than the blasts from patients without resurgence. Materials and Methods Patients and G-CSF Administration AML was diagnosed according to the French- American-British (FAB) criteria [ ], and was classified as MO-M7. Fourteen AML patients who received G-CSF (Filgrastim, Kirin, Japan) for severe infection after chemotherapy at our hospital were enrolled in this study (G-CSF group), and 16 clinically comparable patients with AML who did not receive G-CSF were also included as the control group. The clinical characteristics of the patients are shown in Table I. Table I. Clinical characteristics of the patients No. evaluable 14 (9) 16(11) Mean age i SD 54.9 k [rangel [ [ Sex male 7 (6) 7 (5) female 7 (3) 9 (6) FAB classification MO 1(1) M1 7 (4) 7 (5) M2 1(1) 3 (3) M3 2 (0) 4 (1) M7 3 (3) 1(1) Stage newly diagnosed 7 (4) 6 (2) 1st relapsed 2 (2) refractory* 4 (4) 5 (5) complete remission 2 (0) 3 (0) Chemotherapy B-DMP 7 (2) 6 (2) B-EM 3 (3) 2 (2) LD-AC 2 (2) HD-AC 1(1) 2 (1) V P I6 - A C l(1) l(1) AC CBDCA-AC 0 1(1) 3 (3) 0 Numbers in parentheses show the patients with leukemic resurgence. *Refractoriness was defined according to Hiddernann er al. [33]. Abbreviations: B-DMP, behenoyl-cytosine arabinoside, daunorubicin, 6-mercaptopurine, and prednisolone; B-EM, behenoyl-cytosine arabinoside, etoposide, and mitoxantrone; LD-AC, low-dose cytosine arabinoside; HD-AC, high-dose cytosine arabinoside; VP 16-AC, etoposide and cytosine arabinoside; CBDCA-AC, carboplatin and cytosine arabinoside; AC, cytosine arabinoside.

3 Usuki et al. 649 Leukemic resurgence was defined as an increase of the absolute leukemic blast count in the peripheral blood or an increase of the percentage of leukemic blasts in the bone marrow. The G-CSF group included nine patients (64%) with leukemic resurgence (Table 11), and there were 11 patients with resurgence in the control group. Except in the patients in complete remission, bone marrow examination prior to chemotherapy always showed over 90% blasts. In the G-CSF group, G-CSF was administered S.C. at a daily dose of 75 pg (eight patients), 150 pg (one patient), or 300 pg (five patients) when severe infection resistant to antibiotics developed as a result of chemotherapy. If there was leukemic resurgence, G-CSF was immediately stopped and chemotherapy was continued. Preparation of Leukemic Blasts Leukemic blasts were obtained by sternal aspiration at the time of diagnosis in the patients in complete remission and prior to chemotherapy in the newly diagnosed, relapsed or refractory cases. Leukemic blasts were isolated from the bone marrow by Ficoll density centrifugation. For the colony-forming assay, contaminating T cells were then removed by rosetting with sheep erythrocytes followed by centrifugation through Ficoll, as described elsewhere [25]. Cell Proliferation Isolated leukemic cells were adjusted to 5 x 105/ml in RPMl 1640 medium supplemented with 10% fetal calf serum (FCS) in the absence or presence of 1, 10 or 100 ng/ml of G-CSF. After incubation for four days in 5% C02 at 37 C, the number of cells was counted [26]. [ HI Thymidine Incorporation Assay Cells were cultured as described above. After incubation for 72 h, 0.2 pci of [)H]thymidine was added, and the cells were incubated for a further 15 h. Then the incorporated [ Hlthymidine was counted using a betaplate (Pharmacia, Uppsala, Sweden) [26]. Leukemic Colony Assay Isolated leukemic blast cells were plated in 35-mm plastic Petri dishes at 4 x lo5 cells/ml. Culture was done in 0.8% methylcellulose with 20% FCS and 0, 1, 10 or 100 ng/ml of G-CSF in a-modified Eagle s medium. After seven days, colonies containing more than 20 cells were counted using an inverted microscope, as described elsewhere [25]. G-CSF Radioreceptor Assay Using mutant recombinant human G-CSF, the receptor binding assay was performed as described previously [27]. The mutant protein ([Tyr, Tyr l recombinant human G-CSF), the biological activity of which was equal to that of G-CSF, was prepared by the replacement of threonine-1 and leucine-3 of G-CSF with tyrosine [27]. This mutant G-CSF was radioiodinated using the lactoperoxidase/gludose oxidase method [28] with the Enzymobead reagent (Bio- Rad Laboratories, Richmond, CA). Isolated leukemic blast cells (2.5 x lo6) were incubated for 3 h at 15 C in 200 pl of binding buffer containing labeled mutant G-CSF. The cells were then layered onto 350 p1 of FCS in tapered 0.5 ml flexible plastic centrifuge tubes and centrifuged for 1 min at 1500 g. The tubes were cut off just above the pellet, and the radioactivity of the pellet was measured using Packard Crystal 5454 automated gamma counter. Specific binding was determined as the difference between the amount of radioactivity bound in the absence of unlabeled G-CSF and the amount bound in the presence of excess unlabeled G-CSF. All experiments were conducted in duplicate. The number of binding sites (Bmax) and the dissociation constant (Kd) were obtained by Scatchard analysis [29]. Iinmunophenotyic Analysis Immunophenotyping of isolated leukemic blasts was performed by fluorescenceactivated cell sorter (FACS) analysis using the following monoclonal antibodies: OKT9 (CD7 l), anti-hpca-2 (CD34), glycophorin A, Leu-5b (CD2), Leu-9 (CD7), J5 (CDlO), My7(CD13), Leu-12 (CD19), My9 (CD33), anti-hla-dr, and phycoerythrin-conjugated G-CSF (G-CSFR; R&D systems). Results Cell Proliferation in Liquid Culture Leukemic blasts from 10 of 14 patients in the G-CSF group were incubated in liquid culture in the presence of various concentrations of G-CSF. As shown in Figure IA, after the addition of G-CSF a concentration-dependent increase of

4 Table 11. Details of the patients with leukemic resurgence - during - G-CSF administration chemotherapy of G- before after before after classification stage treatment 1 51 M MO newly B-DMP diagnosed F M1 newly VP16-AC diagnosed F M1 refractory LD-AC M M1 refractory B-EM M M1 refractory CBDCA-AC F M2 relapsed B-EM M M7 newly HD-AC diagnosed? 8 34 M M7 newly B-DMP diagnosed n -. J 9 58 M M7 refractory B-EM *Day of leukemic resurgence: the first day of leukemic regrowth during G-CSF administration.?in these two patients (cases 1 and 8), leukemic resurgence was also confirmed by bone marrow examination. In case no. 1, the percentage of marrow blasts before and after G-CSF treatment was 60% and 74%, respectively, while the percentages were 8.6% and 16.2% in case no. 8. v, R 5 3 z 9. a (P

5 Usuki et al cell numbers was observed in three of the five patients with leukemic resurgence (cases 2, 4 and 5), which was defined as an increase of the absolute blast count in the peripheral blood or an increase of the percentage of blasts in the bone marrow. However, a similar increase was also shown by blasts from three of the five patients without leukemic resurgence (cases 1 I, 13 and 14) (Fig. 1B). The most marked in vitro stimulation of cell proliferation by G-CSF was observed in case 11, even though this patient showed no leukemic resurgence during G-CSF administration. In addition, leukemic blasts from 10 of the 16 patients in the control group were analyzed. In vitro stimulation of blast proliferation was observed in three of the seven patients with leukemic resurgence and two of the three patients without it (data not shown). [3H]thymidine Incorporation [3H] thymidine incorporation by leukemic blasts was assessed in 14 patients from the G-CSF group and 16 patients from the control group. G-CSF dose-dependently stimulated [3H]thymidine incorporation by the leukemic blasts from seven of the nine patients with leukemic resurgence during G-CSF administration (78%; Fig. 1C) as well as the blasts from all five patients without leukemic resurgence (100%; Fig. 1D) in the G-CSF group. Such stimulation of [7H]thymidine incorporation was also seen with blasts from 10 of the 11 patients with leukemic resurgence (91%) as well as all five patients without leukemic resurgence (100%) in the control group (data not shown). After the addition of G-CSF, dose-dependent stimulation of [3H]thymidine incorporation was seen in the blasts from two of the three patients with acute megakaryoblastic leukemia (M7) in the G-CSF group (cases 7 and 9; Fig. 1C) and the blasts from one M7 patient in the control group (data not shown). Leukemic Colony Formation In the G-CSF group, blasts from five of the nine patients with leukemic resurgence during G-CSF administration (Fig. 1E) and three of the five patients without it (Fig. 1F) showed dose-dependent stimulation of leukemic colony formation by G-CSF. The same stimulation was also found for blasts from 8 of the 11 patients with leukemic resurgence and four of the five without it in the control group (data not shown). G-CSF Receptor Leukemic blasts from 12 patients in the G-CSF group and 10 patients in the control group were subjected to the G-CSF radioreceptor assay. A representative experiment is shown in Figure 2. Labeled, modified G-CSF showed dose-dependent specific binding, and nonspecific binding was less than 5% of the total binding in this case. Scatchard analysis revealed a single class of high-affinity binding sites (Kd: 63 pm; 520 sites per cell). The Kd values of blasts from all patients ranged from 32 to 130 pm (mean 77 pm), and the number of binding sites for G-CSF varied from 32 to 6300 per cell (mean 1600). Receptors were all in a single class expression. In the G-CSF group, the G-CSF receptors expressed by blasts from patients showing resurgence had a significantly higher affinity than those of blasts from patients without resurgence (mean Kd: 55 pm versus 101 pm, respectively; both n = 6; p = ; Student s t-test). The G-CSF receptor affinity of blasts in the control group was also significantly higher when leukemic resurgence occurred than when it did not (63 pm versus 96 pm; n = 6 and 4, respectively; p = ; Student s t-test). In the G-CSF group, the number of G-CSF receptors expressed per cell was significantly lower when leukemic resurgence occurred than when it did not (200 per cell versus 3400 per cell, respectively; both n = 6; p = ; Student s t-test). In the control group, the number of G-CSF receptors was also significantly lower when leukemic resurgence occurred than when it did not (260 per cell versus 3030 per cell, respectively; p = ; Student s t-test). The blasts of four patients with acute megakaryoblastic leukemia (M7) had only a few G-CSF receptors (32-73 per cell) with a high affinity (Kd: pm) and G-CSFstimulated [3H]thymidine incorporation by leukemic cells from three of these four patients, as described above. These findings suggest that the leukemic cells of some M7 patients have functional G-CSF receptors. Blast Phenotype Immunophenotypic analysis of the leukemic blasts in all patients from the G-CSF group was done using a FACScan and monoclonal antibodies (for CD2, CD7, CD10, CD13, CD19, CD33, CD34, CD7 1, HLA-DR, and glycophorin A) as

6 652 G-CSF in Acute Myeloid Leukemia 0-CSF concentration (nglrnl) 50 G-CSF concrnhatlon (nglrnl) 0 10 % s5 - i e a 0.5 f F c T 0: ; 1'6 1:o G-CSF concntratlon (ng/rnl) Fig. 1. In vitro effect of G-CSF on the growth of leukemic blasts. The in vitro effect of G-CSF on the growth of leukemic blasts from patients with (A, C, and E) or without (B, D, and F) leukemic resurgence during G-CSF administration was determined by assaying cell numbers in liquid culture (A and B), as well as by [3H]thymidine incorporation (C and D) and colony formation (E and F) in the presence of the indicated concentrations of G-CSF. In A, C, and E, the symbols are as follows: 0 = case no. 1,.=no. 2,.= no. 3, A=no. 4, A = no. 5,o=no. 6,@ = no. 7, V = no. 8, and V = no. 9 (+ indicates case nos. 7-9 in E). In B, D, and F, 0 = case no. 10, = no. 11, A = no. 12, 0 = no. 13, and A no. 14. Data are shown as the mean f SD for triplicate determinations. well as marker-conjugated G-CSF. There were no significant differences between the blasts of patients showing leukemic resurgence during G-CSF administration and those from patients without resurgence (data not shown). Discussion Since G-CSF promotes the growth of leukemic cells in vitro to a variable extent [15-171, it has been suggested that the in vivo influence of this cytokine on leukemic blasts could be predicted by in vitro assays. We therefore explored whether leukemic resurgence during G-CSF administration was related to the in vitro growth response of leukemic cells to this cytokine. We found that the frequency of leukemic resurgence among patients whose blasts showed concentration-dependent proliferation after addition of G-CSF to cultures was similar to that among patients whose blasts did not. In addition, immunophenotypic analysis of leukemic blasts showed no significant differences between cells from patients with and without leukemic resurgence during G-CSF administration. In brief, leukemic cell proliferation in vivo was not correlated with the results of in vitro studies determining blast cell numbers, [3H]thymidine incorporation, leukemic colony formation and phenotype.

7 Usuki et al. 653 s- 4 U a m E Y - U a m Total binding Nonspecific binding o.loh lzsi-[tyrl,tyr3]rhqcsf (pm) 0.10 ht for AML was not correlated with the in vitro responsiveness of leukemic blasts to this Bound lzsl-[tyrl,tyflrhgcsf (pm) Fig. 2. Binding of '251- [Tyr', Tyr3] recombinant human G-CSF to acute myeloid leukemia blasts in case 2. (A) Binding data (B) Scatchard analysis of specific binding. G-CSF receptors were demonstrated on the blast cells of all investigated patients with AML included in this study. Scatchard analysis revealed a slight variation in affinity of a single class of G-CSF receptors (Kd: pm) and considerable variation in the level of expression ( per cell). Similar variations in G-CSF receptor affinity and sites of AML blasts have also been demonstrated by Bludel et al. (Kd: pm; per cell) [30] and Motoji et al. (Kd: pm; per cell) [31]. Bludel et al. reported that mature granulocytes expressed more G-CSF receptors with a slightly less affinity (Kd: pm; per cell) than those on AML cells [30]. Since AML blasts can differentiate into mature granulocytes in vivo [32], this variation might reflect heterogeneous differentiation of leukemic blast population. In both the control and G-CSF groups, the blasts of the patients with leukemic resurgence had G-CSF receptors with a significantly higher affinity than the blasts from patients without resurgence, but expressed significantly fewer receptors. However, Motoji et al. reported that more G-CSF receptors lead to more active proliferation of blast cells in response to G-CSF in vitro [31]. This discrepancy is consistent with our finding that in vivo leukemic resurgence during G-CSF administration was not correlated with in vitro response of blasts to G-CSF. In this study we explored the possibility of predicting the leukemic resurgence after G-CSF treatment after chemotherapy by using G-CSF radioreceptor assay. Our results suggest that AML whose blasts have less G-CSF receptors with a higher affinity is refractory to chemotherapy and that use of this cytokine is safe in patients whose blasts have more G-CSF receptors with a lower affinity. In summary, the leukemic resurgence during G-CSF administration after chemotherapy cytokine or with blast phenotyping data. Leukemic resurgence tended to occur in patients whose blasts had fewer G-CSF receptors with a higher affinity irrespective of whether patients received G-CSF. In addition, the leukemic resurgence during G-CSF treatment did not occur when patients were in complete remission. References 1 Foon KA, Gale RR. Therapy of acute myelogenous leukemia. Blood Rev 1992;6: Reiffers J, Maraninchi D, Rigal-Huguet F et al. Does more intensive treatment cure more patients with acute myeloid leukemia? Semin Hematol 1991 ;28: Morstyn G, Souza LM, Keech J et al. Effect of granulocyte colony-stimulating factor on neutropenia induced by cytotoxic chemotherapy. Lancet 1988; 1: Morstyn G, Campbell L, Lieschke G et al. Treatment of chemotherapy-induced neutropenia by subcutaneously administered granulocyte colony-stimulating factor with optimization of dose and durat.ion of therapy. J Clin Oncol 1989;7: Pettengell R, Gurney H, Radford JA et al. Granulocyte colony-stimulating factor to prevent dose-limiting neutropenia in non-hodgkin's lymphoma: a randomized controlled trial. Blood 1992;SO: Bronchud MH, Scarffe JH, Thatcher N et al. Phase 1/11 study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. Br J Cancer 1987;56:

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