Bone Marrow Findings after Treatment with ...

HEMATOPATHOLOGY Original Article

Bone Marrow Findings after Treatment with Recombinant Human Interleukin-3 STEPHAN FALK, M.D., GERNOT SEIPELT, M.D., ARNOLD GANSER, M.D., OLIVER G. OTTMANN, M.D., DIETER HOELZER, M.D., HANS J. STUTTE, M.D., AND KLAUS HUBNER, M.D.

In a phase I/I I study, bone marrow biopsy specimens and aspirates of 20 patients with malignant tumors but normal bone marrow (n = 6), bone marrow failure resulting from chemotherapy (n = 4), myelodysplastic syndrome (n = 5), and aplastic anemia (n = 5) were evaluated before and after patients were treated with recombinant human interleukin-3 (rhIL-3). This cytokine proved to be an effective hematopoietic growth factor with only mild side effects. The rhIL-3 treatment led to increased overall bone marrow cellularity with trilinear stimulation of hematopoietic cells, except in most patients with aplastic anemia. In all patients, significant eosinophilia and, in some instances,

bone marrow fibrosis developed. In addition to the increase in the number of circulating neutrophilic granulocytes, platelets, and reticulocytes, an increase of peripheral blood monocytes and lymphocytes was observed. The histologic and cytologic findings support the concept that rhIL-3 stimulates the proliferation and differentiation of pluripotent hematopoietic progenitor cells. It appears to be a safe and efficient therapeutic modality in patients with bone marrow failure. Additional clinical studies are needed to determine which patients will profit most from rhIL-3 treatment. (Key words: Bone marrow; Cytopenia; Hematopoiesis; Interleukin-3) Am J Clin Pathol 1991;95:355-362

Interleukin-3 (IL-3), a glycoprotein hormone, has recently become available in large quantities as a recombinant human reagent (rhIL-3) through the use of recombinant DNA techniques.1,2 IL-3 stimulates the proliferation and differentiation of pluripotent hematopoietic stem cells and of committed progenitor cells of granulocyte/macrophage, erythroid, eosinophilic, basophilic, and megakaryocyte lineage.3"5 Moreover, this hematopoietic growth factor enhances effector cell functions in eosinophils and monocytes.6 IL-3 possesses a broader range of activities than those of other hematopoietic growth factors, notably granulocyte-macrophage-colony-stimulating factor (GMCSF), and appears to stimulate very early progenitor cells.3'5'7,8' Several preclinical studies have demonstrated a stimulatory effect of IL-3 on myelopoiesis, erythropoiesis, and thrombopoiesis. 9 "" The current study was conducted as a phase I/II trial in patients with advanced cancer or bone marrow failure resulting from cytotoxic

chemotherapy, myelodysplastic syndrome, or aplastic anemia. One of its objectives was to analyze the bone marrow changes seen morphologically after rhIL-3 treatment and to document the hematopoietic effects of rhlL3 administration by quantitative methods. MATERIALS AND METHODS

Twenty patients (6 women and 14 men with a mean age of 54 years; range, 19-80 years) who were treated with rhIL-3 as part of a phase I/II investigation were studied. The study protocol was approved by the ethics committee of the University of Frankfurt, and informed written consent was obtained from the patients before rhIL-3 administration. Clinical data on the patients are shown in Table 1. Six with advanced malignant tumors (MT group), of whom three had received chemotherapy, had normal hematopoiesis. Four patients were treated with rhIL-3 because of secondary bone marrow failure after prolonged chemotherapy for malignant tumors (MTC group). The median interval between the last course of chemotherapy From the Departments of Pathology and Internal Medicine, Division and the onset of rhIL-3 treatment was 16 weeks (range, of Hematology. University of Frankfurt, Germany. 3-52 weeks). Four of these patients had previously received GM-CSF for bone marrow failure 7 weeks to 6 Received March 2, 1990; received revised manuscript and accepted for publication August 16, 1990. months (median, 16 weeks) before the administration of Address reprint requests to Dr. Falk: Department of Pathology, UnirhIL-3. Five patients had myelodysplastic syndrome as versity of Frankfurt, Theodor-Stern-Kai 7, D-6000 Frankfurt 70, Germany. diagnosed by bone marrow biopsy and cytologic exami355

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HEMATOPATHOLOGY Original Article TABLE 1. PATIENT CHARACTERISTICS Mean Bone Marrow Cellularity

Transfiusions Requirements Age (yr)/ Sex

Diagnosis

37/M 51/F 51/M 67/M 63/M 53/M

Hemangiopericytoma Mesothelioma Renal cell ca Immunocytoma Bile duct ca Renal cell ca

Cx, GM-CSF

63/M 74/M 33/M 66/F

Plasmacytoma NHL(CC) Hodgkin's disease Myeloproliferative syndrome

71/F 48/F 80/F 62/F

RA (5q karyotype) RA (5q karyotype) RA + myelofibrosis RAEB

67/M

RAEB

53/M

AA

Fll

52/M

AA

F18

36/M

AA

FI9 F23

19/M 23/M

AA AA

No. MT group F4 F41 F6 F8 F20 F21 MTC group Fl F2 F44 F46 MDS group F12 F13 F15 F29 F30 AA group F9

Previous Therapy

RBCs

Platelets

%

Before

After rhlL-3

% Increase

rhIL-3 Dose (ng/m'/d)

Vinblastine Steroids

-

-

21 60 57.7 21.8 48.1 28.3

64 71.8 56.7 61.6 58.6 64.9

205 20 -2 183 22 129

60 60 125 250 500 500

Cx, steroids Cx, Rx lCx, Rx Cx, GM-CSF

+ + + +

+ + +

31.7 30 12.4 8.8

38 47 18.8 12.2

20 57 52 39

60 60 60 125

+ + + +

+

43.6 46.7 45 33.2

73 68.3 53.4 45.8

67 46 19 38

250 250 500 500

+

+

9.4

326

500

+

+

13.8

6.1

-56

250

+

+

3

7.9

163

250

—

+

16.7

18.6

11

500

+

+ +

6.9 5.1

32.8 17

275 233

500 500

GM-CSF Steroids, androgens

Steroids, ATG, GM-CSF Steroids, ATG, CSA Steroids, androgens Steroids Steroids, ATG

+

40

MT = malignant tumor, normal hematopoiesis: MTC = malignant tumor, chemotherapy with secondary hematopoietic failure: MDS = myelodysplasia syndrome: AA = aplastic anemia: CML = chronic myeloid leukemia: NHL = non-Hodgkin's lymphoma: CC = centrocyte lymphoma:

RA = refractory anemia: RAEB = refractory anemia with excess of blasts {Frcnch-AmcricanBritish classification): GM-CSF = granulocyte/macrophage colony-stimulating factor ATG = antithymocyte-globulin: CSA = cyclosporin A: RBCs = red blood cells: CX = cytotoxic chemotherapy: RX = radiotherapy.

nation (MDS group). They had peripheral cytopenias and required frequent transfusions of red blood cells and/or platelets. Five patients were included in the trial because of biopsy-proven severe aplastic anemia of unknown origin (AA group). These patients had received immunosuppressive therapy (prednisone, antithymocyte globulin, and/or cyclosporine) without lasting hematologic improvement. The interval between the cessation of immunosuppressive therapy and rhIL-3 treatment was at least four weeks, during which time only supportive transfusions were given. All patients received purified pyrogen- and endotoxinfree rhIL-3 with a specific activity of more than 1 X 107 units/mg protein according to a bone marrow proliferation assay. The recombinant protein was expressed in yeast and obtained from Immunex Corporation/Beringwerke AG (Seattle, WA; Marburg, Germany). 12 The doses used ranged from 60 to 500 ^g/m 2 /day and were administered as subcutaneous bolus injections for 15 days. Regular monitoring of the patients included repeated complete

histories, physical examinations, full blood counts, chemical profiles, and urinalysis, as well as electrocardiograms and chest roentgenograms after completion of the treatment cycle. In addition, bone marrow biopsy specimens were obtained immediately before and after completion of therapy. Bone marrow smears were stained with a modified Giemsa (Pappenheim) stain, whereas biopsy specimens were decalcified and embedded in paraffin. Two- to four-micron paraffin sections were stained with hematoxylin and eosin, Giemsa, periodic acid-Schiff, Perls' iron, and Gomori's silver stain for reticulin fibers. All aspirates and biopsy specimens were coded and examined by two independent observers. In addition to morphologic analysis of the specimens, bone marrow cellularity was evaluated by a point-counting planimetric method with the use of a 100-point graticule at a final magnification of 160X. The relative amount of hematopoietic tissue, fat cells, bone, and vascular structures was calculated from the mean point counts obtained in 15 fields for each biopsy specimen.

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Bone Marrowafter rhIL-3 TABLE 2 . BONE M A R R O W DIFFERENTIAL COUNT IN PATIENTS WITH PRESERVED HEMATOPOIETIC FUNCTION (MT GROUP)

Patient No.

Pre

Myeloblasts Promyelocytes Myelocytes Metamyelocytes Band forms Segmented neutrophils Eosinophils Basophils Lymphocytes Monocytes M/E ratio

0 2 16 13 14 6 2 1 5 3 1.7

F8

F6

F4 Post

Post

Pre

Pre

Post

0 3 14 10 9

0 2 5 22 11

0 9 19 15 12

0 1 6 9 10

1 5 6 12 20

13 9 1 5 1 3.60

11 5 1 7 0 2.00

24 9 2 5 1 2.00

14 2 0 28 1 4.13

5 16 1 9 1 2.20

RESULTS The changes in bone marrow morphologic characteristics and peripheral blood counts are summarized in Tables 2 and 3. MT Group Before rhIL-3 treatment, bone marrow biopsy specimens and aspirates of the six patients of the MT group were unremarkable except for osteoporotic changes in three patients (patients F8, F20, and F41) and moderate

F41

F21

F20 Pre

Post

Pre

Post

0 5 7 8 8

0 9 14 9 8

0 4 7 7 9

0 4 11 14 9

7 8 1 15 2 1.29

14 6 0 6 0 1.12

19 10 0 4 0 3.13

Pre 0 6 9 14 8 6 5 0 9 3 1.23

14 11 1 2 2 2.40

Post 0 8 10 10 9 5 13 0 7 0 1.45

to marked hemosiderosis in patients F4, F20, F21, and F41. In addition, patient F8 exhibited a small nodular infiltrate composed of lymphoid cells that was interpreted as reactive. Mean bone marrow cellularity (BMC) was normal in all patients (39.6 ± 18%, mean ± SD; Fig. 1). After rhIL-3 treatment, BMC increased to a mean of 62.9 ± 5.4%. Accordingly, the bone marrow biopsy specimens showed hyperplasia of all three hematopoietic cell lines and a shift to the left with increased numbers of "immature" (i.e., hypolobated) megakaryocytes, promyelocytes, and myelocytes (Fig. 2, Table 2). Myeloblasts were

TABLE 3. CHANGES IN THE PERIPHERAL BLOOD DURING TREATMENT WITH rhIL-3 Leukocyttes* Patient No. MT group F4 F6 F8 F20 F21 F41 MTC group Fl F2 F44 F46 MDS group F12 F13 F15 F29 F30 AA group F9 Fll F18 F19 F23 ' X109/L ( X I O V M U -

Neutrophils*

Lymphocytes*

Platelets*

Reticulocytes \

Pre

Post

Pre

Post

Pre

Post

Pre

Post

Pre

Post

8.7 9.6 14.4 5.2 6.5 5.8

13.4 10.7 25.3 11.1 25.1 8.7

7.48 7.58 9.22 3.80 4.23 4.12

11.26 7.81 15.08 7.88 15.31 5.63

0.56 0.55 4.46 0.62 1.88 1.22

1.91 2.4 8.66 1.51 5.27 2.35

382 587 332 166 436 225

476 653 468 440 515 386

1.45 0.49 0.80 0.82 1.57 1.02

2.58 1.86 1.77 1.46 4.19 1.77

0.8 4.4 1.8 1.5

1.9 13.4 5.6 2.6

0.16 1.19 0.72 0.30

0.63 2.28 1.01 0.65

0.51 2.64 0.94 1.22

1.22 10.45 3.98 1.85

7 18 17 4

100 185 NA 66

0.10 0.70 0.10 0.40

0.20 0.80 0.20 0.80

3.3 3.0 3.7 1.4 1.5

13.2 5.0 5.8 4.5 2.5

2.04 1.59 1.89 0.3 0.2

10.82 1.75 3.81 1.39 0.62

1.19 1.20 1.41 1.21 1.18

2.14 1.68 1.41 1.66 1.18

188 137 50 7 5

260 NA 86 22 31

0.04 0 0 0.06 1.07

0.21 0.56 0.07 0.14 NA

1.2 1.3 1.9 1.2 2.9

2.7 2.2 4.1 1.8 4.4

0.35 0.47 0.59 0.31 0.35

1.38 0.74 1.85 0.74 1.38

0.71 0.75 1.06 0.82 0.71

1.20 1.12 1.72 1.18 1.20

1 20 12 5 5

33 NA 28 NA NA

0.39 0.25 0.98 0.21 0.22

0.49 0.62 0.67 0.23 0.43

t Corrected reticulocyte count (%). NA = not available.

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HEMATOPATHOLOGY Article turned to baseline levels over a period of two to three weeks. MTC Group

FIG. 1. Slightly hypocellular bone marrow from a patient with carcinoma of the bile duct (MT group) before rhIL-3 treatment. Hematoxylin and eosin (X260).

not detected. The myeloid/erythroid ratio increased from 1.92 ± 0.42 before therapy to 2.46 ± 0.29 after therapy with rhIL-3. Significant eosinophilia was present in all cases (baseline mean, 8.1%, range, 3-17.7%; post-IL-3 mean, 14.6%, range, 8.3-18.9%. Basophils remained normal. The morphologic characteristics of the hematopoietic cells appeared normal; specifically, there were no changes indicative of myelodysplasia. However, an increase of reticulin fibers occurred in patient F8. These bone marrow changes were paralleled by a dose-dependent increase in peripheral reticulocytes (up to 2.6-fold), platelets (up to 2.6-fold), and neutrophil counts (up to 3.6-fold). The increase in reticulocyte counts was delayed and occurred after day 10 of treatment. Platelet counts continued to increase for one week after rhIL-3 therapy and slowly re-

Before rhIL-3 treatment, the bone marrow was hypoplastic in four patients with bone marrow failure secondary to cytotoxic chemotherapy and/or radiation therapy for malignant tumors. In two patients a slight to moderate infiltration by centrocytic lymphoma and plasmacytoma, respectively, was observed (patients Fl and F46). The patient with a long-standing plasmacytoma also exhibited a moderate increase in reticulin fiber density (Fig. 3). After rhIL-3 treatment, mean BMC increased in all four patients. Cytologic abnormalities of the hematopoietic cells could not be detected, and the myeloid/erythroid ratio was preserved. Myeloblasts were not detected. The differentiation ratio of mature to immature myeloid cells (i.e., the ratio of myelocytes plus metamyelocytes plus granulocytes to myeloblasts plus promyelocytes) also was not affected by rhIL-3 therapy. However, bone marrow fibrosis was observed in two patients (Fig. 4). Peripheral blood leukocyte counts after treatment increased to more than 2,000/jtL in all but one patient. In addition, platelet counts increased 1.3-to 14.2-fold (mean, sixfold), allowing for discontinuation of platelet transfusions. Although an increase of reticulocyte counts (mean, twofold) was observed, this increase did not reduce red blood cell transfusion requirements in three of the four patients. After completion of rhIL-3 therapy, two of three patients did not require platelet transfusions any longer (mean followup, ten months).

AJ.C.P. March 1991


FALK ET AL. after rhIL-3 Bone

FIG. 4. Treatment with rhIL-3 led to an increase of bone marrow cellularity but also to marked fibrosis. Same patient as in Figure 3. Hematoxylin and eosin (X260).

MDS Group Four of the five patients with myelodysplastic syndromes (see Table 1 for further subclassification according to the French-American-British [FAB] cooperative group criteria for myelodysplastic syndromes) had normocellular bone marrow, whereas one with refractory anemia with excess blasts (RAEB) exhibited hypocellularity in the pretreatment bone marrow biopsy (Fig. 5). However, in all MDS cases dysplastic features such as nuclear anomalies, with fragmented or accessory nuclei in both erythroid and myeloid precursors, as well as hypolobulated megakaryocytes, were present. Two patients exhibited focally increased myeloblasts, warranting a diagnosis of RAEB.

FIG. 5. Myelodysplastic syndrome with hypoplasia of hematopoietic cells prior to rhIL-3 treatment (MDS group). Note the abnormal megakaryocytes chracteristic of MDS. Hematoxylin and eosin (X260).

359

Administration of rhIL-3 led to an increase in BMC in all patients, which was accompanied by an increase in the degree of fibrosis, however (Fig. 6). Although the bone marrow exhibited pronounced eosinophilia in all cases, significant basophilia was observed in only two patients with the 5q- karyotype. Except in one of these, who exhibited a significant shift of the myeloid/erythroid ratio, necessitating additional red blood cell transfusions, the myeloid/erythroid ratios and the differentiation ratios remained largely unchanged. Percentages of circulating blasts were less than 1 % in all cases. The cytomorphologic characteristics of the hematopoietic cells usually did not change (i.e., the dysplastic features persisted during and after rhIL-3 therapy). After rhIL-3 therapy, the total number of leukocytes (i.e. neutrophils, eosinophils, basophils, lymphocytes, and monocytes) in the peripheral blood increased significantly. Neutrophil counts increased 1.1- to 5.3-fold. The response of platelet and reticulocyte counts was variable: in most patients the degree of anemia remained unchanged, whereas one patient (patient F13) showed a significant reduction of transfusion requirements. The two patients with RAEB who had severe thrombocytopenia exhibited an increase in peripheral platelets, allowing discontinuation of platelet transfusions for two months. AA Group Before rhIL-3 treatment, the bone marrow in the five patients with aplastic anemia exhibited severe depletion of hematopoietic cells with only rare remaining foci of erythropoiesis. In addition, there was marrow edema, siderosis, and plasmacytosis in all cases. Dyshematopoiesis or bone marrow fibrosis was not detectable (Fig. 7). After

FIG. 6. After rhlL-3 treatment, the bone marrow of the same patient is hypercellular and fibrotic. Hematoxylin and eosin (X260).

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HEMATOPATHOLOGY

Original Article high-dose rhIL-3 therapy, granulocyte precursors and a small number of megakaryocytes were observed in addition to erythropoietic foci. Moreover, numerous eosinophils were present. This was also reflected in the bone marrow smears, which, in contrast to the findings before rhIL-3 administration, contained increased numbers of morphologically normal promyelocytes and myelocytes as well as some megakaryocytes, albeit with a very low myeloid/erythroid ratio. One patient (patient F19) showed a fourfold increase in mean BMC (Fig. 8), but there was no effect on the peripheral blood count. Patient F9 showed a transient increase of platelets with a peak count of 31,000/JUL on day 32. In all other patients there was no change in the number of platelets. This also held true for reticulocyte counts: although they reached levels greater than 1%, the transfusion requirements remained unchanged. However, peripheral neutrophils (2.3- to 3.9fold increase), peripheral eosinophils (3.6- to 19-fold increase), and monocytes (1.4- to 5.5-fold increase) were augmented. It should be mentioned that side effects of rhIl-3 in all patients, regardless of the underlying condition, were few and generally mild. They included low-grade fever, headaches, and flushing. These side effects did not necessitate cessation of treatment in any case. DISCUSSION Infections and hemorrhage are the most frequent causes of death in patients with bone marrow failure resulting from chemotherapy or radiation therapy, myelodysplastic syndrome, or aplastic anemia. Treatment has been largely restricted to supportive red blood cell and platelet trans-

FlG. 7. Severely hypoplastic bone marrow showing only small foci of erythropoiesis with some admixed plasma cells in aplastic anemia (AA group) before rhIL-3 treatment. Hematoxylin and eosin (X260).

FIG. 8. After rhIL-3, hematopoietic cells, including myeloid precursors, are markedly increased. Same patient as in Figure 7. Hematoxylin and eosin (X260).

fusions as well as antibiotic therapy. The advent of recombinant hematopoietic growth factors such as GM-CSF or G-CSF has fundamentally altered this situation, because numerous studies have shown that the administration of these substances leads to elevated total leukocyte counts in patients with bone marrow failure.13"15 However, these hematopoietic growth factors usually do not stimulate megakaryocyte proliferation and maturation, 16 and their effects partly result from mobilization of mature blood cells from the bone marrow or the marginal pool.17 Therefore, a multilineage hematopoietic growth factor such as IL-3, which exerts its action at the level of pluripotent and committed hematopoietic precursor cells4'7,816 and has been shown to cause trilinear hyperplasia of hematopoietic marrow in small rodents, 18 would be preferable to G-CSF and GM-CSF. 19 The results of the current study offer morphologic evidence of a multilineage response of hematopoietic cells to rhIL-3 in patients with preserved bone marrow function (MT group) and in patients with bone marrow failure and long-lasting cytopenias secondary to cytotoxic chemotherapy or myelodysplasia (MTC and MDS group). The results in patients with aplastic anemia (AA group) are less convincing. Although a meaningful statistical evaluation of each treatment group was precluded by the small number of patients studied, there was a convincing overall increase of BMC in those treated with rhIL-3. After completion of rhIL-3 treatment, several patients in this study did not require erythrocyte and/or platelet transfusions during a mean follow-up time of 10-12 months. In all MT cases, rhIL-3-induced hyperplasia of the bone marrow was paralleled by a delayed increase of peripheral blood cell counts. This delay, the long duration of the stimulatory effects, and the concomitant increase in pe-

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361

FALK ET AL. Bone Marrow after rhIL-3 ripheral lymphocytes support the assumption that the action of IL-3 as a growth factor is centered on early pluripotent progenitor cells rather than on morphologically recognizable myeloid cells in the bone marrow.20 The effect of rhIL-3 was most notable in the increase of peripheral platelet and leukocyte counts; in fact, in some patients the increase in platelet numbers was paralleled by a striking increase in the number of megakaryocytes in the bone marrow. This is in keeping with earlier results that have shown that rhIL-3 is a very effective stimulus for megakaryocyte differentiation.21 However, because other patients exhibited a constant number of megakaryocytes that in some instances were hypolobated, the platelet increase could also result from an increase in the number of platelets produced by individual megakaryocytes.

but may again be ascribed to a stimulation of bone marrow fibroblasts. The disappointing hematologic response to rhIL-3 treatment in patients with aplastic anemia could result from a significant depletion of hematopoietic stem cells, a disturbed hematopoietic microenvironment with failure of cytokine production, and/or immunologic mechanisms.26 It is interesting that some clinical trials with other hematopoietic growth factors (i.e., GM-CSF and G-CSF) have had similarly transient results.12,27 Clearly, additional studies are needed to determine the role of rhIL-3 as part of treatment regimens for aplastic anemia that may involve immunosuppression and long-term administration of combinations of recombinant cytokines.9

Except in patients with myelodysplastic syndromes, hematopoietic cells (see below) appeared morphologically normal without any dysplastic features after treatment with rhIL-3. The increase in the number of eosinophils both in the bone marrow and in the peripheral blood is not surprising and could be predicted from preclinical studies.22 In contrast, it is surprising that basophilia only occurred in the two patients with 5q- syndrome, of which the significance is largely unknown. 2223 The patients with significant basophilia did not exhibit clinical signs of increased histamine release. Prior cytotoxic chemotherapy and/or increases in bone marrow fiber content before rhIL-3 therapy seem to predispose patients to the development of significant bone marrow fibrosis, as observed in 4 of 19 patients in this subgroup. This effect might result from stimulation of bone marrow fibroblasts by rhIL-3. In patients with myelodysplastic syndromes, the morphologic and clinical response to rhIL-3 differed from the changes observed in patients with preserved bone marrow function. Specifically, the increase in granulocyte counts was variable, and the increase in platelets as well as reticulocytes was not very pronounced although it was of clinical importance, resulting in a reduction of transfusion requirements.24 These features are largely attributable to the intrinsic stem cell defects in patients with MDS. In the MDS patients, the proliferative effect of this cytokine on immature progenitor cells is apparently balanced by the induction of differentiation,25 which corresponds to the presence of maturing, albeit dysmorphic, hematopoietic cells in the bone marrow of these patients. None of the MDS patients included in this study has had his or her disease progress to overt leukemia (mean follow-up, 12 months). Additional studies are needed to define criteria that help to identify those patients with MDS who run the risk of overt leukemic transformation after rhlL3 treatment. As in the MTC group, the pathogenesis of the rhIL-3-related bone marrow fibrosis in MDS is unclear

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