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Treatment of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor: A preliminary report
Treatment of Myelodysplastic Syndromes with Recombinant Human Granulocyte Colony-Stimulating Factor: A Preliminary Report YUKIOKOBAYASHI, M.D., TETSURO~KABE, M.D., D.M.s., KEIYAOZAWA, M.D., D.M.s., SHIGERU CHIBA, M.D., MASAYUKI HINO, M.D., KOHEIMIYAZONO,M.D., AKIOURABE,M.D., D.M.s., FUMIMAROTAKAKU, M.D.,
D.M.S.
Tokyo,
Japan
he myelodysplastic syndromes result from the gradual expansion of an abnormal hemopoietic T stem cell clone and are characterized by pancytopen-
PURPOSE: The expansion of an abnormal hemopoietic stem cell line is responsible for the myelodysplastic syndromes, which are characterized by pancytopenias, often resulting in lethal infections. Cloned granulocyte colony-stimulating factor (GCSF) was recently shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro. The aim of our study was to examine the effects of recombinant human G-CSF in patients with myelodysplastic syndromes. PATIENTS AND METHODS: Four patients with myelodysplastic syndromes and one patient with smoldering acute myelogenous leukemia following the occurrence of a myelodysplastic syndrome received recombinant human G-CSF by intravenous infusion for six days. Patients received different dosage levels (50 to 1,600 rcg/m2). RESULTS: A response was seen in all patients, with an increase in both immature myeloid cells in the bone marrow and mature granulocytes in the peripheral blood. The dose levels that could stimulate granulocytopoiesis differed among patients. CONCLUSION: These results suggest that, at least in some cases of myelodysplastic syndromes, granulocytopenia can be improved by G-CSF, although it still remains to be determined whether the increase in the number of granulocytes is due to the differentiation and maturation of the myelodysplastic clone or restoration.of a residual normal clone.
ias, often resulting in lethal infections [l--3]. Human granulocyte colony-stimulating factor (G-CSF) preferentially stimulates the formation of Day 7 granulocyte colonies in human marrow cultures [4-61. G-CSF was recently cloned [7-91 and shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro [lo]. We report herein on five patients treated with recombinant G-CSF. PATIENTS AND METHODS Permission for the trial was obtained from the Committee on Clinical Investigation at the University of Tokyo. Informed patient consent was also obtained. Five patients were diagnosed as having or having had myelodysplastic syndromes according to the FrenchAmerican-British Cooperative Group criteria [2]. The patients’ characteristics are shown in Table I. The patients ranged in age from 22 to 61 years old. Durations from the diagnosis to the initiation of therapy ranged from five to 18 months. None of the patients required platelet transfusions. Only Patient 5 had received periodic red blood cell transfusions. A male patient (Patient 5), diagnosed as having myelodysplastic syndrome, refractory anemia with excess of blasts 18 months before, had received low-dose cytosine arabinoside and BHAC-DMP therapy [ll] without improvement. The count of the blasts in the bone marrow of this patient had been almost stable but gradually increased to more than 30 percent, which should be classified as acute myelogenous leukemia. However, considering the slowly progressive course, this patient was also included into the protocol. No infection was evident in the patients. They were ambulatory with sufficient hepatic and renal function (serum glutamic oxaloacetic transaminase less than 66 U/liter; serum glutamic pyruvic transaminase less than 100 U/liter; creatinine less than 2.0 mg/dl). Patients received a daily 30-minute intravenous infusion of G-CSF (Kirin Brewery Co. Ltd., Tokyo, Japan). The material was human G-CSF [7] produced by Escherichia coli. The specific activity was 1 x lo8 U/ mg protein. Since dosage levels of 50 or 100 pg/m2/day for six consecutive days were previously shown to cause granulocytosis in normal healthy volunteers and the safety of G-CSF had been fully ascertained (Azuma J, Takaku F: Phase I study of recombinant granulocyte colony-stimulating factor in normal healthy volunteers; submitted for publication), initial dosage levels of 50 or 100 Kg/m2 were chosen for safety in the first trial. If changes in the hematologic parameters
From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. This work was supported in part by a grantin-aid for cancer research from the Ministry of Health and Welfare in Japan. This study was presented in part at the General Meeting of the American Federation of Clinical Research held in Washington, D.C., in April 1988, as “Differentiation therapy of myelodysplastic syndrome by recombinant granulocyte colony-stimulating factor”. Requests for reprints should be addressed to Yukio Kobayashi, M.D., the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan. Manuscript submitted August 17, i988, and accepted in revised form December 1, 1988.
178
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1989
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Volume
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G-CSF THERAPY
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1
TABLE I Patients Treated with G-CSF Patient Number
Age Sex (years)
Diagnosis
i
61 22 50 63
MDS, MDS, MDS, MDS,
5
56 M
:
M F M M
RA RA RAE8 RA
MDS +AML
White Blood Cell Counts OW
Hemoglobin WW
Platelets (X 104/mm3)
Dx-TX (months)
7.1 7.0 9.9 8.0
2,200 1,600 1,400 7,600
1.0 1.2 5.2 2.6
:i
6.8
1,000
0.7
18
Previous Treatment
Kaorotype .
Transfusion* Transfusion* Transfusion* (-)
E
46XY, normal 46Xx, normal 46XY, normal 45XY,-5,-22 +mar, del(7) 46XY, normal
Transfusiont LDAraC BHAC-DMP
Dx-TX = interval from diagnosis to treatment with G-CSF; MDS = myelodysplastic syndrome; RA = refractory anemia; RAEB = refractory anemia with excess of blasts: AML = acute myelogenous leukemia: LDAraC = low-dose cytosine arabinoside; BHAC-DMP = combination chemotherapy using behonyl cytosine
arabinoside, daunorubicin. 6-mercaptopurine. and prednisolone. * Red blood cell transfusion only when symptoms appeared (3, 2, and 6 packs) for Patients 1, 2, and 3, respectively t Periodic red cell transfusion.
such as the number of granulocytes, immature myeloid cells in the bone marrow (by morphology), and precursors (colony-forming units-culture) were minimal, the dosage level was increased. When a significant increase was observed, therapy was discontinued, and these parameters were further observed.
RESULTS A rapid increase in peripheral leukocytes was observed after a single injection of G-CSF (Figure l), an increase that began 4.5 hours after injection. These changes were observed in all 5 patients on Day 1. The peak occurred between 8.5 and 12.5 hours after injection. This pattern was almost the same as that in norma1 healthy subjects [la]. The response after six consecutive days of treatment was also evaluated. The chosen dose level of 50 or 100 pg/m2 could effectively raise the counts in normal healthy volunteers 1121,which is also equivalent to the dose level (3 pug/kg) that is reported to be able to increase the number of absolute neutrophils on Days 6 and 7 by 3.1 to 4.0 times [13]. This dose level (50 or 100
,ug/m2) also increased the number of granulocytes in two patients (3 and 4) after six days of treatment. However, in the other three patients (Patients 1, 2, and 5), the increased counts of leukocytes and granulocytes were not sustained shortly after injection, and the dose level was increased. A dose level of 400 pg/m2/ day cauied an increase in the granulocyte count in Patients 1 and 2. Patient 1, for example, did not respond to treatment with 50 pglm2/day of G-CSF. At the increased dose level, the patient achieved an almost normal level of granulocytes (Figure 2 and Table II). The dose level for Patient 5 was further increased to 1,600 pglm21day, which is still less than the maximal tolerated dose determined by Gabrilove et al [13]. At this dose, peripheral leukocyte counts remained higher than at the pretreatment level. In virtually all the cases, granulocytes that appeared after therapy had the same morphologic abnormalities as those before therapy. For example, in Patient 1, granulocytes with pseudo-Pelger-Hiiet anomaly, which could also be observed before treatment, increased in number. Changes in the morphology of the
2b 5c
Figure I. Change in leukocyte numbers shortly after single injection of G-CSF. Percents against initial counts are indicated. Case la: percents after injection at the dose level of 50 @g/m’; lb: 400 wg/m2; 2a: 50 pg/m2; 2b: 400 pg/m2; 5a: 50 pg/m2; 56: 400 pg/m2; 5c: 1,600 pg/m2.
0
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1989
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Figure 2. Changes
/ KOBAYASHI
ET AL
in numbers of total leukocytes and granulocytes during G-CSF treatment. Open circles indicate the number OT total circles indicate the number of granulocytes. Numbers below each figure show percent of blasts and myeloid series in the
leukocytes. Closed bone marrow.
TABLE II Hematologic
SYNDROMES
Data before and after Treatment
Patient Number
White Blood Cells (/mm31
Peripheral Granulocytes (/mm31
Blood Hemoglobin k/W
Platelets (/mm31
Bone Marrow M Blasts* (percent of blasts)
1 Before400treatmentf After kg/m*+
1,800 4,600
750 2,100
5”:;
8,000 5,000
25.2 48.4
1.2 2.4
Before400treatment After pg/m*
1,700 4,000
360 2,300
5.8 5.6
57,000 9,000
18.6 66.0
0.4 0.8
Before100 treatment After Kg/m*
1,300 4,900
390 2,500
9.8
8.9
55,000
49,000
17.2 50.0
14.4 16.4
Before100 treatment After fig/m*
9,200 13,700
6,800 10,400
7.4 6.2
53,000 64,000
44.1 82.0
0.0 0.0
1,000 1,800
4::
6.6 7.5
5,000 2,000
3.6 6.4
67.2 25.2
I)L
3
4
5 Before1,600 treatment After pg/m*
Hyeloid series. Percent of total promyelocytes + myelocytes + mature granulocytes. Before treatment: samples were taken on Day 0. After treatment: samples were taken on Day 7.
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dysplastic myeloid precursors and mature cells such as the size and amount of granulations and poly-lobe counts were not observed. The granulocyte count declined promptly upon termination of the therapy and returned to pretreatment levels within three days after discontinuation. All the patients showed an increase in myeloid precursors such as myelocytes and promyelocytes. There was no evidence of an increase in blasts; rather, the number of the blasts decreased. There was an increase in the myeloid-to-erythroid ratio in the bone marrow. Bone marrow specimens obtained from the patients demonstrated an increase in the cellularity with the expansion of myeloid series. The colony count assayed as colony-forming units-culture [14] was 0 or extremely lower than normal levels before therapy in all the cases. A few colonies did appear after therapy. In Patients 1 and 4, small clusters consisting of less than 30 cells were observed (Table III). There was no change in the number, size, or maturation of the megakaryocytes and erythroid series. As far as we could determine, colony-forming units-erythroid did not change significantly (Patient 1, from 55 f 10 to 76 f 12; Patient 2 from 17 f 3 to 14 f 3; all others, 0). Serum biochemical data stayed within normal limits. The level of alkaline phosphatase increased by 5 to 20 percent during the course of the treatment; it remained within the normal range. During the course of treatment, there were no subjective toxicities.
COMMENTS We used G-CSF in five patients in an attempt to improve the granulocytopenias in myelodysplastic syndromes. To date, G-CSF has been reported to act as a physiologic granulopoietin in pre-clinical studies; it also increases the number of granulocytes in mice and reduces the granulocytopenic period induced by chemotherapy and irradiation [15-171. Results of phase I and II studies in cancer patients have already been reported. Gablilove et al [18], Morstyn et al [19], and Bronchud et al [20] had demonstrated an increase in circulating neutrophils when G-CSF was injected into cancer patients as well as a shortening of the period of the granulocytopenia induced by chemotherapy. Furthermore, G-CSF is known to repair functional abnormalities of neutrophils of patients with myelodysplastic syndromes [21]. Therefore, G-CSF is one of the most promising agents for treating granulocytopenia in patients with myelodysplastic syndromes. All the patients had a rapid response to this agent at the relatively higher dose level, showing an increase in the number of granulocytes within a week. The response to G-CSF appeared to be a combination of several events. The rapid increase in granulocyte counts beginning 4.5 hours after injection suggests that this response is a part of a demargination and/or movement of mature cells from the bone marrow. In fact, at lower doses (Patients 1 and 2), the rapid increase in counts after injection was not accompanied by an increase in immature bone marrow myeloid cells; the granulocyte counts returned to the pretreatment level before injection on the following day. The rise was not sustained by these lower doses, and rapidly declined to the pretreatment level after discontinuation of treatment. At higher doses, the number of myeloid, cells also
FOR MYELODYSPLASTIC
SYNDROMES
/ KOBAYASHI
ET AL
TABLE III Bone Marrow CFU-C Assay
Patient Number
Before Therapy Colt Glut
After 50* rel m* Cal CIU
After 400 k&m* Cal CIU
I
5
0
0
0
0
0
0
* 50 or 100 pg/m*. t Col = number of normal CFU-C colonies; Clu = number of abnormal clusters. A colony assay was performed using a single-layer soft-agar culture system [14]. Bone marrow aspirate was collected and mononuclear layer cells (5 X 104/plate) were plated in 0.5 ml aliquots of alpha medium containing 20 percent fetal bovine serum, 0.3 percent agar, and 10 percent giant cell tumorconditioned medium [5] as a source for colony-stimulation factors.
increased in the bone marrow. This response appears to be a second proliferation at the progenitor level. Although the colony count remained at the baseline level in each patient except for Patient 4, the appearance after therapy might be considered as if some change has occurred at the progenitor level. In fact, we failed again to demonstrate the colonies several months after therapy. We can explain the increase in myeloid series by either or both of two mechanisms. One is the differentiation and maturation of the dysplastic clone; the other is the restoration of the normal clone. The observation that the peripheral granulocytes were still morphologically abnormal clusters suggests that the former is likely [22] but not conclusive. We cannot determine the origin of the colony without cytogenetic analysis. Mature neutrocytes that appeared after therapy must also be studied to determine their origin by such means as restriction fragment lengths polymorphisms. We observed no changes in hematologic parameters other than myeloid series. Another potential CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), has an advantage in that patients will have improvements in red blood cells and platelets as well as leukocytes [23]. Comparison of G-CSF with GMCSF must be contemplated in the future, as well as the use of a combination of G-CSF and GM-CSF. We limited treatment in these five patients to only six days because we planned to use G-CSF as a supportive agent only after infection had occurred. We must determine if G-CSF can actually prevent infections, change the course of myelodysplastic syndromes, or both. Moreover, we must evaluate the risk of leukemic conversion. To date, during the observation period (five to 12+ months), no such problems have been encountered. A trial of injection for longer periods including self-injections is needed. Such an ongoing trial is being conducted by Negrin et al [24]. Our patients were treated in a pilot study, and the sample size is small and the result is preliminary. Dose-limiting toxicity and an optimal schedule still remain to be determined. Previous reports suggest that the dose can be increased to 800 pg/m2/day with minimal toxicity [18-201. Again, more experience, with longer follow-up periods, is needed to determine whether patients with myelodysplastic syndromes can actually benefit from this treatment. February
1989
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/ KOBAYASHI
ET AL 13. Gabrilove JL, Jakubowski A, Fran K, et a/: Phase I study of granulocyte colonystimulatingfactor in patients with transitional cell carcinoma of the urethelium. J Clin Invest 1988; 82: 1454-1461. 14. Ozawa K. Sato N, Urabe A, Takaku F: Modulation of myelopoiesis in vitro by retinoic acid. Biochem Biophys Res Commun 1984; 123: 128-132. 15. Fujisawa M, Kobayashi Y, Okabe T, Takaku F, Komatsu Y. ltoh S: Recombinant human granulocyte colony-stimulating factor induces granulocytosis in viva Jpn J Cancer Res 1986; 77: 866-869. 16. Shimamura M, KobayashiY, Yuo A, eta/; Effects of human recombinantgranulocyte colony-stimulating factor on hematopoietic injury in mice induced by 5. fluorouracil. Blood 1987; 69: 353-355. 17. Kobayashi Y, Okabe T, Urabe A, Suzuki N, Takaku F: Human recombinant granulocyte colony-stimulating factor produced by Escherichia co/i shortens the period of granulocytopenia induced by irradiation in mice. Jpn J Cancer Res 1987; 78: 763-766. 18.Gabrilove JL, Jakubowski A, Sher H, et al: A study of granulocyte colonystimulating factor in cancer patients at risk for chemotherapy-induced neutropenia. N Engl J Med 1988; 318: 1414-1422. 19. Morstyn G, Cambell L, Duehsen U, eta/:Granulocyte colony-stimulating factor (G-CSF): abrogation of cytotoxic chemotherapy induced neutropenia. Lancet 1988; I: 667-671. 20. Bronchud MH, Scarffe JH, Thatcher N, et al: Phase l/II study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. Brit J Cancer 1987; 56: 809-813. 21.Yuo A, Kitagawa S, Okabe T, et al: Recombinant human granulocyte colonystimulating factor repairs the abnormalities of neutrophils in patients with myelodysplastic syndromes and chronic myelogenous leukemia. Blood 1987; 70: 404411. 22. Messner HA: In vitro growth of leukemia cells. In: Gale RP. Golde DW, eds. Leukemia: recent advances in biology and treatment. Alan R Liss, Inc., New York, 1985; 427-435. 23. Vadham-Raj S, Keating M, LeMaistre A, et al: Effects of recombinant granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 1987; 317: 1545-1552. 24. Negrin RS, Haeuber DH, Nagler A, Souza LM, Greenberg PA: Treatment of myelodysplastic syndromes with recombinant human granulocyte colonystimulating factor (abstr). Exp Hematol 1988; 16: 519.
ACKNOWLEDGMENT We thank Dr. A. Shimosaka
and Dr. T. Kaneko for providing
G-CSF.
REFERENCES 1. Prognosis in myelodysplasia (ed). Lancet 1986; II: 436-437. 2. Bennett JM, Catovsky D, Daniel MT, eta/: Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51: 189-199. 3. Buzaid AC, Garewell HS, Greenberg BR: Management of myelodysplastic syndromes. Am J Med 1986; 80: 1149-1157. 4. Nicola NA, Metcalf D, Johnson GR, Burgess A: Separation of functionally distinct human granulocyte-macrophase colony-stimulating factors. Blood 1979: 54: 614627. 5. Abboud CN, Brennan JK, Barlow GH, Lichtman MA: Hydrophobic adsorption chromatography of colony-stimulating activities and erythroid enhancing activity from the human monocyte-like cell lin. GCT. Blood 1981; 58: 114&l 154. 6. Nicola NA, Begley CG. Metcalf D: Identification of the human analogue of a regulator that induces differentiation in murine leukemic cells. Nature 1985; 314: 625-628. 7. Nagata S, Tsuchiya M, Asano S, et al: Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 1986; 319: 415-418. 8. Komatsu Y, Matsumoto T, Kuga T. et at Cloning of granulocyte colony-stimulating factor cDNA from human macrophages and its expression in Escberichia co/i. Jpn J Cancer Res 1987; 78: 1179-1181. 9. Sachs L: The molecular control of blood cell development. Science 1987; 238: 1374-1379. 10. Souza LM, Boone TC, Gabrilove J. et al: Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 1986; 232: 61-65. 11. Ohno R, Kato Y, Nagura E, eta/: Behenoyl cytosine arabinoside, daunorubicin, C-mercaptopurine, and prednisolone combination therapy for acute myelogenous leukemia in adults and prognostic factors related to remission and survival length. J Clin Oncol 1986; 4: 1740-1747. i2. Kobayashi Y, Okabe T. Uzumaki H, Urabe A, Takaku F: Differentiation therapy of myelodysplastic syndromes by recombinant human granulocyte colony-stimulating factor (abstr). Clin Res 1988; 36: 412A.
182
February
1989
The American
Journal
of Medicine
Volume
86
D.M.S.
Tokyo,
Japan
he myelodysplastic syndromes result from the gradual expansion of an abnormal hemopoietic T stem cell clone and are characterized by pancytopen-
PURPOSE: The expansion of an abnormal hemopoietic stem cell line is responsible for the myelodysplastic syndromes, which are characterized by pancytopenias, often resulting in lethal infections. Cloned granulocyte colony-stimulating factor (GCSF) was recently shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro. The aim of our study was to examine the effects of recombinant human G-CSF in patients with myelodysplastic syndromes. PATIENTS AND METHODS: Four patients with myelodysplastic syndromes and one patient with smoldering acute myelogenous leukemia following the occurrence of a myelodysplastic syndrome received recombinant human G-CSF by intravenous infusion for six days. Patients received different dosage levels (50 to 1,600 rcg/m2). RESULTS: A response was seen in all patients, with an increase in both immature myeloid cells in the bone marrow and mature granulocytes in the peripheral blood. The dose levels that could stimulate granulocytopoiesis differed among patients. CONCLUSION: These results suggest that, at least in some cases of myelodysplastic syndromes, granulocytopenia can be improved by G-CSF, although it still remains to be determined whether the increase in the number of granulocytes is due to the differentiation and maturation of the myelodysplastic clone or restoration.of a residual normal clone.
ias, often resulting in lethal infections [l--3]. Human granulocyte colony-stimulating factor (G-CSF) preferentially stimulates the formation of Day 7 granulocyte colonies in human marrow cultures [4-61. G-CSF was recently cloned [7-91 and shown to enhance the growth and differentiation of normal granulocyte progenitor cells in vitro [lo]. We report herein on five patients treated with recombinant G-CSF. PATIENTS AND METHODS Permission for the trial was obtained from the Committee on Clinical Investigation at the University of Tokyo. Informed patient consent was also obtained. Five patients were diagnosed as having or having had myelodysplastic syndromes according to the FrenchAmerican-British Cooperative Group criteria [2]. The patients’ characteristics are shown in Table I. The patients ranged in age from 22 to 61 years old. Durations from the diagnosis to the initiation of therapy ranged from five to 18 months. None of the patients required platelet transfusions. Only Patient 5 had received periodic red blood cell transfusions. A male patient (Patient 5), diagnosed as having myelodysplastic syndrome, refractory anemia with excess of blasts 18 months before, had received low-dose cytosine arabinoside and BHAC-DMP therapy [ll] without improvement. The count of the blasts in the bone marrow of this patient had been almost stable but gradually increased to more than 30 percent, which should be classified as acute myelogenous leukemia. However, considering the slowly progressive course, this patient was also included into the protocol. No infection was evident in the patients. They were ambulatory with sufficient hepatic and renal function (serum glutamic oxaloacetic transaminase less than 66 U/liter; serum glutamic pyruvic transaminase less than 100 U/liter; creatinine less than 2.0 mg/dl). Patients received a daily 30-minute intravenous infusion of G-CSF (Kirin Brewery Co. Ltd., Tokyo, Japan). The material was human G-CSF [7] produced by Escherichia coli. The specific activity was 1 x lo8 U/ mg protein. Since dosage levels of 50 or 100 pg/m2/day for six consecutive days were previously shown to cause granulocytosis in normal healthy volunteers and the safety of G-CSF had been fully ascertained (Azuma J, Takaku F: Phase I study of recombinant granulocyte colony-stimulating factor in normal healthy volunteers; submitted for publication), initial dosage levels of 50 or 100 Kg/m2 were chosen for safety in the first trial. If changes in the hematologic parameters
From the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. This work was supported in part by a grantin-aid for cancer research from the Ministry of Health and Welfare in Japan. This study was presented in part at the General Meeting of the American Federation of Clinical Research held in Washington, D.C., in April 1988, as “Differentiation therapy of myelodysplastic syndrome by recombinant granulocyte colony-stimulating factor”. Requests for reprints should be addressed to Yukio Kobayashi, M.D., the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan. Manuscript submitted August 17, i988, and accepted in revised form December 1, 1988.
178
February
1989
The American
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Volume
86
G-CSF THERAPY
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1
TABLE I Patients Treated with G-CSF Patient Number
Age Sex (years)
Diagnosis
i
61 22 50 63
MDS, MDS, MDS, MDS,
5
56 M
:
M F M M
RA RA RAE8 RA
MDS +AML
White Blood Cell Counts OW
Hemoglobin WW
Platelets (X 104/mm3)
Dx-TX (months)
7.1 7.0 9.9 8.0
2,200 1,600 1,400 7,600
1.0 1.2 5.2 2.6
:i
6.8
1,000
0.7
18
Previous Treatment
Kaorotype .
Transfusion* Transfusion* Transfusion* (-)
E
46XY, normal 46Xx, normal 46XY, normal 45XY,-5,-22 +mar, del(7) 46XY, normal
Transfusiont LDAraC BHAC-DMP
Dx-TX = interval from diagnosis to treatment with G-CSF; MDS = myelodysplastic syndrome; RA = refractory anemia; RAEB = refractory anemia with excess of blasts: AML = acute myelogenous leukemia: LDAraC = low-dose cytosine arabinoside; BHAC-DMP = combination chemotherapy using behonyl cytosine
arabinoside, daunorubicin. 6-mercaptopurine. and prednisolone. * Red blood cell transfusion only when symptoms appeared (3, 2, and 6 packs) for Patients 1, 2, and 3, respectively t Periodic red cell transfusion.
such as the number of granulocytes, immature myeloid cells in the bone marrow (by morphology), and precursors (colony-forming units-culture) were minimal, the dosage level was increased. When a significant increase was observed, therapy was discontinued, and these parameters were further observed.
RESULTS A rapid increase in peripheral leukocytes was observed after a single injection of G-CSF (Figure l), an increase that began 4.5 hours after injection. These changes were observed in all 5 patients on Day 1. The peak occurred between 8.5 and 12.5 hours after injection. This pattern was almost the same as that in norma1 healthy subjects [la]. The response after six consecutive days of treatment was also evaluated. The chosen dose level of 50 or 100 pg/m2 could effectively raise the counts in normal healthy volunteers 1121,which is also equivalent to the dose level (3 pug/kg) that is reported to be able to increase the number of absolute neutrophils on Days 6 and 7 by 3.1 to 4.0 times [13]. This dose level (50 or 100
,ug/m2) also increased the number of granulocytes in two patients (3 and 4) after six days of treatment. However, in the other three patients (Patients 1, 2, and 5), the increased counts of leukocytes and granulocytes were not sustained shortly after injection, and the dose level was increased. A dose level of 400 pg/m2/ day cauied an increase in the granulocyte count in Patients 1 and 2. Patient 1, for example, did not respond to treatment with 50 pglm2/day of G-CSF. At the increased dose level, the patient achieved an almost normal level of granulocytes (Figure 2 and Table II). The dose level for Patient 5 was further increased to 1,600 pglm21day, which is still less than the maximal tolerated dose determined by Gabrilove et al [13]. At this dose, peripheral leukocyte counts remained higher than at the pretreatment level. In virtually all the cases, granulocytes that appeared after therapy had the same morphologic abnormalities as those before therapy. For example, in Patient 1, granulocytes with pseudo-Pelger-Hiiet anomaly, which could also be observed before treatment, increased in number. Changes in the morphology of the
2b 5c
Figure I. Change in leukocyte numbers shortly after single injection of G-CSF. Percents against initial counts are indicated. Case la: percents after injection at the dose level of 50 @g/m’; lb: 400 wg/m2; 2a: 50 pg/m2; 2b: 400 pg/m2; 5a: 50 pg/m2; 56: 400 pg/m2; 5c: 1,600 pg/m2.
0
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G-CSF THERAPY
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Figure 2. Changes
/ KOBAYASHI
ET AL
in numbers of total leukocytes and granulocytes during G-CSF treatment. Open circles indicate the number OT total circles indicate the number of granulocytes. Numbers below each figure show percent of blasts and myeloid series in the
leukocytes. Closed bone marrow.
TABLE II Hematologic
SYNDROMES
Data before and after Treatment
Patient Number
White Blood Cells (/mm31
Peripheral Granulocytes (/mm31
Blood Hemoglobin k/W
Platelets (/mm31
Bone Marrow M Blasts* (percent of blasts)
1 Before400treatmentf After kg/m*+
1,800 4,600
750 2,100
5”:;
8,000 5,000
25.2 48.4
1.2 2.4
Before400treatment After pg/m*
1,700 4,000
360 2,300
5.8 5.6
57,000 9,000
18.6 66.0
0.4 0.8
Before100 treatment After Kg/m*
1,300 4,900
390 2,500
9.8
8.9
55,000
49,000
17.2 50.0
14.4 16.4
Before100 treatment After fig/m*
9,200 13,700
6,800 10,400
7.4 6.2
53,000 64,000
44.1 82.0
0.0 0.0
1,000 1,800
4::
6.6 7.5
5,000 2,000
3.6 6.4
67.2 25.2
I)L
3
4
5 Before1,600 treatment After pg/m*
Hyeloid series. Percent of total promyelocytes + myelocytes + mature granulocytes. Before treatment: samples were taken on Day 0. After treatment: samples were taken on Day 7.
180
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1989 The American
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G-CSF THERAPY
dysplastic myeloid precursors and mature cells such as the size and amount of granulations and poly-lobe counts were not observed. The granulocyte count declined promptly upon termination of the therapy and returned to pretreatment levels within three days after discontinuation. All the patients showed an increase in myeloid precursors such as myelocytes and promyelocytes. There was no evidence of an increase in blasts; rather, the number of the blasts decreased. There was an increase in the myeloid-to-erythroid ratio in the bone marrow. Bone marrow specimens obtained from the patients demonstrated an increase in the cellularity with the expansion of myeloid series. The colony count assayed as colony-forming units-culture [14] was 0 or extremely lower than normal levels before therapy in all the cases. A few colonies did appear after therapy. In Patients 1 and 4, small clusters consisting of less than 30 cells were observed (Table III). There was no change in the number, size, or maturation of the megakaryocytes and erythroid series. As far as we could determine, colony-forming units-erythroid did not change significantly (Patient 1, from 55 f 10 to 76 f 12; Patient 2 from 17 f 3 to 14 f 3; all others, 0). Serum biochemical data stayed within normal limits. The level of alkaline phosphatase increased by 5 to 20 percent during the course of the treatment; it remained within the normal range. During the course of treatment, there were no subjective toxicities.
COMMENTS We used G-CSF in five patients in an attempt to improve the granulocytopenias in myelodysplastic syndromes. To date, G-CSF has been reported to act as a physiologic granulopoietin in pre-clinical studies; it also increases the number of granulocytes in mice and reduces the granulocytopenic period induced by chemotherapy and irradiation [15-171. Results of phase I and II studies in cancer patients have already been reported. Gablilove et al [18], Morstyn et al [19], and Bronchud et al [20] had demonstrated an increase in circulating neutrophils when G-CSF was injected into cancer patients as well as a shortening of the period of the granulocytopenia induced by chemotherapy. Furthermore, G-CSF is known to repair functional abnormalities of neutrophils of patients with myelodysplastic syndromes [21]. Therefore, G-CSF is one of the most promising agents for treating granulocytopenia in patients with myelodysplastic syndromes. All the patients had a rapid response to this agent at the relatively higher dose level, showing an increase in the number of granulocytes within a week. The response to G-CSF appeared to be a combination of several events. The rapid increase in granulocyte counts beginning 4.5 hours after injection suggests that this response is a part of a demargination and/or movement of mature cells from the bone marrow. In fact, at lower doses (Patients 1 and 2), the rapid increase in counts after injection was not accompanied by an increase in immature bone marrow myeloid cells; the granulocyte counts returned to the pretreatment level before injection on the following day. The rise was not sustained by these lower doses, and rapidly declined to the pretreatment level after discontinuation of treatment. At higher doses, the number of myeloid, cells also
FOR MYELODYSPLASTIC
SYNDROMES
/ KOBAYASHI
ET AL
TABLE III Bone Marrow CFU-C Assay
Patient Number
Before Therapy Colt Glut
After 50* rel m* Cal CIU
After 400 k&m* Cal CIU
I
5
0
0
0
0
0
0
* 50 or 100 pg/m*. t Col = number of normal CFU-C colonies; Clu = number of abnormal clusters. A colony assay was performed using a single-layer soft-agar culture system [14]. Bone marrow aspirate was collected and mononuclear layer cells (5 X 104/plate) were plated in 0.5 ml aliquots of alpha medium containing 20 percent fetal bovine serum, 0.3 percent agar, and 10 percent giant cell tumorconditioned medium [5] as a source for colony-stimulation factors.
increased in the bone marrow. This response appears to be a second proliferation at the progenitor level. Although the colony count remained at the baseline level in each patient except for Patient 4, the appearance after therapy might be considered as if some change has occurred at the progenitor level. In fact, we failed again to demonstrate the colonies several months after therapy. We can explain the increase in myeloid series by either or both of two mechanisms. One is the differentiation and maturation of the dysplastic clone; the other is the restoration of the normal clone. The observation that the peripheral granulocytes were still morphologically abnormal clusters suggests that the former is likely [22] but not conclusive. We cannot determine the origin of the colony without cytogenetic analysis. Mature neutrocytes that appeared after therapy must also be studied to determine their origin by such means as restriction fragment lengths polymorphisms. We observed no changes in hematologic parameters other than myeloid series. Another potential CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), has an advantage in that patients will have improvements in red blood cells and platelets as well as leukocytes [23]. Comparison of G-CSF with GMCSF must be contemplated in the future, as well as the use of a combination of G-CSF and GM-CSF. We limited treatment in these five patients to only six days because we planned to use G-CSF as a supportive agent only after infection had occurred. We must determine if G-CSF can actually prevent infections, change the course of myelodysplastic syndromes, or both. Moreover, we must evaluate the risk of leukemic conversion. To date, during the observation period (five to 12+ months), no such problems have been encountered. A trial of injection for longer periods including self-injections is needed. Such an ongoing trial is being conducted by Negrin et al [24]. Our patients were treated in a pilot study, and the sample size is small and the result is preliminary. Dose-limiting toxicity and an optimal schedule still remain to be determined. Previous reports suggest that the dose can be increased to 800 pg/m2/day with minimal toxicity [18-201. Again, more experience, with longer follow-up periods, is needed to determine whether patients with myelodysplastic syndromes can actually benefit from this treatment. February
1989
The American
Journal
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181
GCSF
THERAPY
FOR MYELODYSPLASTIC
SYNDROMES
/ KOBAYASHI
ET AL 13. Gabrilove JL, Jakubowski A, Fran K, et a/: Phase I study of granulocyte colonystimulatingfactor in patients with transitional cell carcinoma of the urethelium. J Clin Invest 1988; 82: 1454-1461. 14. Ozawa K. Sato N, Urabe A, Takaku F: Modulation of myelopoiesis in vitro by retinoic acid. Biochem Biophys Res Commun 1984; 123: 128-132. 15. Fujisawa M, Kobayashi Y, Okabe T, Takaku F, Komatsu Y. ltoh S: Recombinant human granulocyte colony-stimulating factor induces granulocytosis in viva Jpn J Cancer Res 1986; 77: 866-869. 16. Shimamura M, KobayashiY, Yuo A, eta/; Effects of human recombinantgranulocyte colony-stimulating factor on hematopoietic injury in mice induced by 5. fluorouracil. Blood 1987; 69: 353-355. 17. Kobayashi Y, Okabe T, Urabe A, Suzuki N, Takaku F: Human recombinant granulocyte colony-stimulating factor produced by Escherichia co/i shortens the period of granulocytopenia induced by irradiation in mice. Jpn J Cancer Res 1987; 78: 763-766. 18.Gabrilove JL, Jakubowski A, Sher H, et al: A study of granulocyte colonystimulating factor in cancer patients at risk for chemotherapy-induced neutropenia. N Engl J Med 1988; 318: 1414-1422. 19. Morstyn G, Cambell L, Duehsen U, eta/:Granulocyte colony-stimulating factor (G-CSF): abrogation of cytotoxic chemotherapy induced neutropenia. Lancet 1988; I: 667-671. 20. Bronchud MH, Scarffe JH, Thatcher N, et al: Phase l/II study of recombinant human granulocyte colony-stimulating factor in patients receiving intensive chemotherapy for small cell lung cancer. Brit J Cancer 1987; 56: 809-813. 21.Yuo A, Kitagawa S, Okabe T, et al: Recombinant human granulocyte colonystimulating factor repairs the abnormalities of neutrophils in patients with myelodysplastic syndromes and chronic myelogenous leukemia. Blood 1987; 70: 404411. 22. Messner HA: In vitro growth of leukemia cells. In: Gale RP. Golde DW, eds. Leukemia: recent advances in biology and treatment. Alan R Liss, Inc., New York, 1985; 427-435. 23. Vadham-Raj S, Keating M, LeMaistre A, et al: Effects of recombinant granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes. N Engl J Med 1987; 317: 1545-1552. 24. Negrin RS, Haeuber DH, Nagler A, Souza LM, Greenberg PA: Treatment of myelodysplastic syndromes with recombinant human granulocyte colonystimulating factor (abstr). Exp Hematol 1988; 16: 519.
ACKNOWLEDGMENT We thank Dr. A. Shimosaka
and Dr. T. Kaneko for providing
G-CSF.
REFERENCES 1. Prognosis in myelodysplasia (ed). Lancet 1986; II: 436-437. 2. Bennett JM, Catovsky D, Daniel MT, eta/: Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 51: 189-199. 3. Buzaid AC, Garewell HS, Greenberg BR: Management of myelodysplastic syndromes. Am J Med 1986; 80: 1149-1157. 4. Nicola NA, Metcalf D, Johnson GR, Burgess A: Separation of functionally distinct human granulocyte-macrophase colony-stimulating factors. Blood 1979: 54: 614627. 5. Abboud CN, Brennan JK, Barlow GH, Lichtman MA: Hydrophobic adsorption chromatography of colony-stimulating activities and erythroid enhancing activity from the human monocyte-like cell lin. GCT. Blood 1981; 58: 114&l 154. 6. Nicola NA, Begley CG. Metcalf D: Identification of the human analogue of a regulator that induces differentiation in murine leukemic cells. Nature 1985; 314: 625-628. 7. Nagata S, Tsuchiya M, Asano S, et al: Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. Nature 1986; 319: 415-418. 8. Komatsu Y, Matsumoto T, Kuga T. et at Cloning of granulocyte colony-stimulating factor cDNA from human macrophages and its expression in Escberichia co/i. Jpn J Cancer Res 1987; 78: 1179-1181. 9. Sachs L: The molecular control of blood cell development. Science 1987; 238: 1374-1379. 10. Souza LM, Boone TC, Gabrilove J. et al: Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 1986; 232: 61-65. 11. Ohno R, Kato Y, Nagura E, eta/: Behenoyl cytosine arabinoside, daunorubicin, C-mercaptopurine, and prednisolone combination therapy for acute myelogenous leukemia in adults and prognostic factors related to remission and survival length. J Clin Oncol 1986; 4: 1740-1747. i2. Kobayashi Y, Okabe T. Uzumaki H, Urabe A, Takaku F: Differentiation therapy of myelodysplastic syndromes by recombinant human granulocyte colony-stimulating factor (abstr). Clin Res 1988; 36: 412A.
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1989
The American
Journal
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Volume
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