Recombinant Human Interleukin 3: Effect on Thrombopoiesis in Patients with Primary and Secondary Hemopoietic Failure

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1 Recombinant Human Interleukin 3: Effect on Thrombopoiesis in Patients with Primary and Secondary Hemopoietic Failure Dieter Hoelzer, Gernot Seipelt, Oliver G. Ottmann, Arnold Ganser Department of Hematology, University of Frankfurt, Frankfurt, Federal Republic of Germany Key Words. Human interleukin 3 Thrombopoiesis Myelopoiesis Clinical trials Aplastic anemia Cancer chemotherapy Bone marrow transplantation Myelodysplastic syndrome Abstract. Yeast-synthesized recombinant human interleukin 3 (rhil-3) has been studied in a phase I/II clinical trial initially in patients with normal hemopoietic function (NH). It then was investigated in patients with cytopenias, particularly prolonged secondary hemopoietic failure after chemotherapy and/or radiotherapy (SHF), bone marrow transplantation (BMT), in myelodysplastic syndromes (MDS) and in aplastic anemia (AA). Dose levels ranged from pg/m2 daily for 5 days administered as S.C. bolus injection. The clinical results demonstrate the capacity of rhil-3 to stimulate myelopoiesis in all patient groups (increase in leukocytes in: NH 2.8-fold, SHF 2.9-fold, BMT 6.7-fold, MDS.8-fold, AA.8-fold) and thrombopoiesis with an increase in platelets in patients with NH (.9-fold) and SHF (2.7-fold), but to a lesser degree in patients with MDS (.7-fold in 5/9 patients) after BMT (increase in /3 patients) and only rarely in AA (increase in /9 patients). A stimulation of erythropoiesis with a rise in reticulocyte counts was observed in 4 of 9 patients with NH, in 6/8 with SHF, in /3 after BMT and 4/9 with AA. However, this reticulocyte increase was not, in most cases, followed by a rise in hemoglobin. Thus, rhil-3 induces a multilineage response and seems to be a promising cytokine to stimulate megakaryopoiesis in patients with thrombocytopenias associated with a variety of underlying diseases. clinical In Vitro and In Vivo Studies Recombinant human interleukin 3 (rhil-3) belongs to a family of glycoprotein hormones responsible for regulating hematopoietic and immune functions, although not necessarily under steady-state conditions [I]. rhil-3 stimulates Correspondencc: Dr. D. Hoelzer, Abteilung fur Hamatologie, Zentrum der Inneren Medizin, Klinikum der Johann Wolfgang Goethe Universitat, Theodor-Stem-Kai 7, D Frankfurt/M 70, FRG. Received July 2, 99 ; accepted for publication July 2, /9 /$2.00/0 OAlphaMed ss

2 Hoelzer/Seipelt/Ottrnann/Ganser 69 Table I. Patient characteristics Diagnosis N Age (yrs) Male/fernale Transfusion dependency Median Range platelets erythrocytes Normal hernopoiesis Secondary hernopoietic failure Bone marrow transplantation Myelodysplastic syndrome Aplastic anemia the proliferation and differentiation of hematopoietic precursor cells [2, 3 and enhances the function of mature myeloid cells such as phagocytosis, antibodydependent cellular cytotoxicity, metabolism of eosinophils but not neutrophils, as well as monocyte cytotoxicity [4-6. The range of activities of rhil-3 is broader than that of the other colony-stimulating factors including granulocytemacrophage colony-stimulating factor (GM-CSF), granulocyte-csf (G-CSF) and macrophage-csf (M-CSF) [7], and in particular, it appears to be a more potent stimulator of megakaqopoiesis [8,9]. clinical studies have demonstrated that rhil-3 effectively stimulates myelopoiesis, erythropoiesis and thrombopoiesis in several murine, rat and nonhuman primate models [ 0-5. Clinical Phase 0-Studies in Humans On the basis of these data and the availability of sufficient quantities of rhil-3 produced by recombinant DNA techniques [20], we have treated patients with normal hemopoiesis having advanced malignancies [ 7, patients with prolonged cytopenias due to radiotherapy and/or chemotherapy [ 7 or bone marrow transplantation for leukemia [2], patients with myelodysplastic syndromes (MDS) [8] or aplastic anemia [9] in phase ID clinical trials. (Patient characteristics given in Table I.) The trial was designed to investigate the: - pharmacokinetics - toxicity and safety profile of rhil-3 - effect on mature circulating blood cells - incidence and proliferation rate of progenitor cells in the bone marrow and peripheral blood

3 70 Clinical Trials with Interleukin-3 I rh-interleukin-3 Fig.. Effect of rhil-3 on the platelet counts in patients with normal hemopoietic function at four different doses of rhil-3 given S.C. for 5 days. * = 60 pg (n = 2); 0 =25pg(n=6);O =250pg(n=5);A=500pg(n=2). Table. Effect of IL-3 treatment on bone marrow progenitor cells of patients with normal hernopoiesis Incidence per lo5 cells Percent in S phase Post Post CFU-gernm 7 i f 2.8 0k6 40i7 BFU-e 82f 6 57 k 8.5 6rt7 36f4 d4, CFU-gm I50 f f 24 i3 26f3 d7, CFU-grn 268 f f 3 3f3 3f3 Treatment Schedule The treatment schedule consisted of dose levels of 30p8/m2, 60pg/mZ, 25,ug/ m', 250 pg/m' and 500 pg/m2 of rhil-3, administered by S.C. bolus injection daily for 5 days. An individual patient was treated on one dose level only. The first dose on day was administered over 5 minutes as an i.v. bolus injection at the dose levels 60 and 25 pg/m2 in order to determine the pharmacokinetics of rhil-3. rhil-3 in Patients with Normal Hemopoietic Function Twenty patients with advanced malignancy, but normal bone marrow function, were treated with rhil-3 to evaluate the safety as well as the hematological effects induced by this cytokine.

4 Hoelzer/Seipelt/Ottmann/Ganser 7 Table. Adverse clinical effects of rhil-3 (n = 46) Adverse Effects WHO Grade Fever Headache Chills Neck rigidity Bone pain Flush Rash Local erythema Weight loss Asthenia Lethargy Vomiting Dyspnea Night sweat Thromboc ytopenia In response to daily S.C. administration of rhil-3, the peripheral blood cell counts rose in a dose-dependent, but delayed manner. An increase in platelet counts was observed in 5 of 8 evaluable patients ranging from.3-fold at 60,ug/m2 to.9-fold at 250 pg/m. The platelet counts sometimes continued to rise for an additional week after discontinuation of rhil-3 treatment before retuming to baseline levels over a period of 2 to 3 weeks. Mean peak leukocyte counts rose.-fold (at a daily dose of 60 pg/m2) to 2.8-fold (at a dose of 500 pg/m2) (Fig. ). The increase in leukocytes was primarily due to a dose-dependent increase in segmented neutrophils, eosinophils and lymphocytes. Circulating neutrophil counts increased dose-dependently up to 2.7-fold at 500 pg/mz. After discontinuation of treatment, neutrophil numbers returned to baseline levels within one to two weeks. Reversible eosinophilia occurred at all dose levels with a 3- to 7-fold increase. Basophil counts also increased up to 5.9-fold at the dose level of 500 pg/m2. Lymphocytes were increased between.9- and 2.4-fold after treatment; both T-helper and -suppressor lymphocytes were affected (for absolute values see [ 7). A stimulation of erythropoiesis with an increase in reticulocyte counts was observed in 4 patients. However, no dose-response relationship was present. In two patients, the increase in reticulocytes resulted in a rise of the hemoglobin level by more than one g/dl. Following treatment with rhil-3, the percentage of actively cycling bone marrow erythroid burst-forming units (BFU-e) and granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-gemm) significantly increased 2.2- and 4-fold, respectively (Table ) [23]. A comparable stimulation

5 72 Clinical Trials with Interleukin-3 Table IV. Secondary hemopoietic failure4hanges in peripheral blood count (cells per nl) after rhil-3 Platelets Post* Total leukocytes posta Neu trophils Post" Reticulocytes Post" "Maximum response during study Median oo Range was also observed for the early and late granulocyte-macrophage colony-forming units (CFU-gm). The incidence of bone marrow CFU-gemm remained constant, whereas the incidence of BFLJ-e and CFU-gm increased, taking into account that the bone marrow cellularity increased.6-fold in patients with normal hemopoiesis [22]. Mean levels of circulating CFU-gemm and CFU-gm were increased after seven days of treatment but returned nearly to baseline levels after 5 days of therapy. In contrast, peripheral blood BFLJ-e were reduced in the majority of patients by treatment with rhil-3 [23]. The histological analysis of bone marrow biopsies obtained prior to and after the treatment cycles demonstrated an increase in cellularity from 39.5 percent to 63.2 percent, along with incre,ases in megakaryocytes and eosinophils but not in basophils [22]. Granulopoiesis was shifted to the left with increased percentages of promyelocytes and myelocytes, while the percentage of lymphocytic elements decreased from.6 to 6.7. However, taking into account the increase in cellularity during treatment with rhil-3, the amount of lymphoid cells in the bone marrow did not change. Toxicity of the treatment schedule was mild. Fever was seen in 70 percent of the patients and was usually more pronounced during the first days of therapy and at higher dosages. Further common side effects included headache, chills and bone pain (Table ). rhil-3 in Patients with Secondary Hemopoietic Failure The characteristics of the patients with prolonged secondary bone marrow failure which were treated with rhil-3 are given in Table I. Bone marrow hypoplasia was due to prolonged chemotherapy and radiotherapy for cancer in

6 ~ %con: ~ Hoelzer/Seipel t/ottmann/ganser 73 Table V. Bone marrow transplantation4hanges in peripheral blood counts after rhil-3 Pt. No. Platelets/nl WBC/nl Neutrophils/nl Lymphocytes/nl Reticulocytes Post Post Post Post Post st cycle ndcycle Table VI. Myelodysplastic syndromes4hanges in peripheral blood count (cells per nl) after rhl-3 Platelets Post" Total leukocytes PosP Neutrophils Post3 Reticulocytes Post" "Maximum response during study Median Range nine patients and additional tumor infiltration of the bone marrow in three of the nine patients as confirmed by bone marrow cytology and histology. In none of the patients was there any evidence of spontaneous hemopoietic recovery. A stimulation of thrombopoiesis with a subsequent sixfold increase in platelet numbers occurred in five of eight evaluable patients, and platelet transfusions could be discontinued permanently in two out of three patients who had been dependent on repeated transfusions. The total leukocyte counts as well as the neutrophil counts rose in all evaluable eight patients in response to rhil-3 (Fig. 2). As compared to patients with normal hemopoietic function, the rise in circulating leukocytes was more delayed, but eventually seven out of eight patients achieved a 3.7-fold increase in neutrophil counts (Table IV). The median time to peak neutrophil counts was 9 days. Reticulocyte counts responded to administration of rhil-3 in six of eight patients. The red blood cell transfusion requirements, however, were unchanged. Bone marrow cellularity also increased in six of eight patients with an increase in the percentage of immature myeloid cells in four of eight patients [22]. There were no increases in bone marrow infiltrating tumor or lymphoma cells.

7 ~~ Hoelzer/Seipelt/O ttmann/ganser 75 Table VII. Aplastic anemia+hanges rhil-3 in peripheral blood count (cells per nl) after Platelets Post Total leukocytes Post4 Neutrophils Post" Reticulocytes Post" "Maximum response during study Median Range 0-35 response in / chromosome 5 (3 RA, RAEB) [8]. Increases in platelet counts, accompanied by a reduced bleeding tendency, were seen in two patients with profound thrombocytopenia allowing a discontinuation of platelet transfusions for more than four months. Pronounced responses of the leukocyte counts to rhil-3 injections were observed with significant increases in the number of total leukocytes, neutrophils, eosinophils, basophils, lymphocytes and monocytes. Neutrophilic granulocytes increased twofold immediately after the end of rhil-3 therapy in all nine patients. After discontinuation of rhil-3 treatment, leukocyte counts gradually decreased to baseline levels. A substantial increase was also observed for eosinophils, basophils and lymphocytes (Table V). The response of reticulocytes was diverse. In a single patient an increase in the hemoglobin level was associated with a transient reduction of transfusion requirements. In contrast, in two patients with 5q syndrome, erythropoiesis was diminished following rhil-3 treatment, resulting transiently in increased transfusion requirements. Reversible increases in circulating blast cells occurred in two patients not exceeding one percent in the differential count. In a further patient, the increase in circulating blast cells was sustained and was accompanied by a rise of blast cells in the bone marrow from 2 percent to 26 percent while on study. Bone marrow samples obtained in all patients prior to and after the treatment cycles demonstrated significant increases in cellularity as well as in the ME-ratio and eosinophils [ 8, 22. Basophilia was induced in the four patients with the 5q syndrome with 4.6- to 6-fold increase. Increases in megakaryocytes occurred in four patients. rhil-3 had no effect on megakaryocyte morphology with persistent hypolobulation of the nucleus in the patients with the 5q-syn-

8 76 Clinical Trials with Interleukin-3 Table VIII. Overall response to interleukin 3 treatment Diagnosis Platelets Neutrophils Reticulocyte Normal 7/20 7/20 6/20 hemopoiesis Hemopoietic 59 failure Bone marrow 3 transplantation Myelodysplas- 79 tic syndromes Aplastic 9 anemia drome. rhil-3 did not lead to a induction of maturation of the myeloid cells. RFLP-analysis showed persistence of monoclonality in one patient (RA 5q-), whereas in another patient (RA) complete polyclonality in the peripheral blood could be demonstrated after rhil-3 while in the bone marrow the hemopoiesis remained monoclonal [27]. rhil-3 in Patients with Aplastic Anemia Nine patients with aplastic anemia, all transfusion-dependent except one, were treated with rhil-3 at daily dosages between 250 pg/m2 (n = 3) and 500 pg/ m2 (n = 6). All except two patients had been pretreated with immunosuppressive agents. Two patients had already been treated with recombinant GM-CSF at six weeks to one year prior to entering the present trial but without any lasting improvements of white blood cell counts [9]. The hematological responses of the nine patients to treatment with rhil-3 were moderate (Table VII). Only one patient showed a transient increase in platelet numbers from I,OOO/pl on day to 3,0OO/p on day 32. The requirement for platelet transfusions transiently decreased in this patient. Platelet counts and transfusion requirements were unchanged in the other eight patients. Neutrophil counts increased at least twofold in five of the nine patients. The peak increase in neutrophil counts (.3- to 3.9-fold) occurred at a median of 2 days with early responses on days 3 and 4 and returned to baseline levels within three weeks after discontinuation of rhil-3 treatment. In four patients the corrected reticulocyte counts slightly increased; transfusion requirements however, were not reduced in any of the patients. Bone marrow samples obtained prior to and after the treatment cycles demonstrated increases in cellularity in three of the patients together with

9 Hoelzer/Seipelt/Ottmann/Ganser 77 increases in eosinophils [22]. In two patients the cellularity actually normalized at the end of rhil-3 treatment without, however, resulting in a substantial increase of peripheral leukocyte counts. Discussion Treatment with rhil-3 clearly induced a multilineage response with an increase in leukocytes, platelets and reticulocytes in patients with normal hemopoietic function (Table VIII). The most important and clinically promising response to rhil-3 was the increase of platelet and reticulocyte counts in patients with prolonged secondary bone marrow failure. rhil-3 treatment in patients with prolonged cytopenias after chemotherapy and/or radiotherapy or bone marrow transplantation resulted in a sustained improvement of bone marrow function and new blood formation as has been described in another study with rhil-3 treatment for 28 days [30]. The response of the platelets and reticulocytes induced by rhil-3 is consistent with the concept that rhil-3 exerts its principle action at the level of multipotent and committed hematopoietic progenitor cells [2,3, 3,32 and not at the level of morphologically recognizable precursor cells in the bone marrow [5]. rhil-3 is a potent stimulating factor of both megakaryocytic and erythroid progenitor cells [32]. The longer lasting effects of rhil-3 on platelet counts differ from those on white blood cell counts and reticulocyte counts, probably due to differences in the level of stimulation and in transit times between the various cell lineages. That the increase in reticulocyte counts did not result in an elevation of the hemoglobin level in the majority of the patients could be due to the short treatment duration of 5 days or the relative lack of a later acting cytokine such as erythropoietin [3]. The increase in platelet counts was dependent on the dose of rhil-3 given, the highest increases occurring most uniformly at 250 &m2. The basis of the delayed response of leukocyte counts to rhil-3, in contrast to a more rapid action of G-CSF or GM-CSF on leukocyte counts, again seems to be the stimulation at the level of the multipotent and lineage committed progenitor cells. This is further supported by our data on the cell cycle status of the progenitor cells CFU-gemm, BFU-e and CFU-gm [23]. The number of circulating progenitor cells did not increase, as was to be expected from monkey studies [ 5. The incidence of bone marrow CFU-gemm remained constant, whereas the incidence of BFU-e and CFU-gm increased slightly, taking into account that the bone marrow cellularity increased.6-fold in patients with normal hematopoietic function [22]. Rapid mobilization of mature cells from the bone marrow within hours after administration of rhil-3 as suggested from studies in rats [ 5 was also observed. In patients with MDS, rhil-3 transiently corrected leukopenia with an increase in bone marrow cellularity and in granulocytic elements resulting in a

10 78 Clinical Trials with Interleukin-3 rise of circulating leukocytes. The leukocyte response to rhil-3 was comparable to that observed in patients with normal hemopoiesis. The increase in platelet and reticulocyte counts induced by rhil-3 was only moderate in comparison to the findings in patients with normal hemopoiesis or with secondary hemopoietic failure. Nevertheless, two out of four profoundly thrombocytopenic transfusion-dependent patients had a clinical benefit from a modest increase of platelet counts in that they did not require platelet transfusions for a period of four months. With regard to its effects in aplastic anemia, treatment with rhil-3 induced a hemopoietic response, albeit moderate, with an increase in leukocytes in five of nine patients. Mobilization of cells from the bone marrow 5 or marginal pool may have accounted for the response in some patients, since the maximum values of neutrophil and lymphocyte counts were reached earlier than in patients with normal hemopoiesis and too early to have resulted from an effect on progenitor cells. The minor response to rhil-3 treatment in aplastic anemia in the majority of patients could be due to a lack or deficiency of IL-3-responsive stem cells. In the patients with marked increases in bone marrow cellularity after rhil-3 treatment, the increases in leukocyte counts were not sustained after the end of treatment, probably indicating that rhil-3 should be administered continuously for prolonged periods in future trials in combination with immunosuppressive therapy. In conclusion, rhll-3 appears to be a potent and well-tolerated multilineage stimulant of thrombopoiesis, leukocytopoiesis and erythropoiesis in man. rhil- 3 may be of considerable value in reducing chemotherapy- and radiotherapyinduced morbidity and mortality or in the treatment of disease-related pancytopenic disorders. References Ihle J, Keller J, Oroszlan S, et al. Biologic properties of homogenous interleukin 3. J Immunol 983;3: Saeland S, Caux C, Favre C, et al. Effects of recombinant human interleukin 3 in CD34-enriched normal hematopoietic progenitors and on myeloblastic leukemia cells. Blood 988;72: Sonoda Y, Yang YC, Wong GG, Clark SC, Ogawa M. Analysis in serum-free culture of the targets of recombinant human hemopoietic growth factors: interleukin- 3 and granulocyte-macrophage colony-stimulating factor are specific for early development stages. Proc Natl Acad Sci 988;85: Rothenberg ME, Owen WF Jr, Silberstein DS, et al. Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3. J Clin Invest 988;8: Lopez AF, Dyson PG, To LB, et al. Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness in differentiation in the neutrophilic myeloid series. Blood 988;72:

11 Hoelzer/Seipelt/Ottmann/Ganser 79 6 Cannistra SA, Vellenga E, Groshek P, Rambaldi A, Griffin JD. Human granulocytemonocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor-dependent mechanism. Blood 988;7: Emerson SG, Yang YC, Clark SC, Long MW. Human recombinant granulocytemacrophage colony-stimulating factors and interleukin-3 have overlapping but distinct hematopoietic activities. J Clin Invest 988;82: Bruno R, Briddell R, Hoffman R. Effect of recombinant and purified hematopoietic growth factors on human megakaryocyte colony formation. Exp Hematol 988; Teramura M, Katahira J, Hoshino S, Motoji T, Oshimi K. Clonal growth of human megakaryocyte progenitors in serum-free cultures: effect of recombinant human interleukin-3. Exp Hematol 988; 6: Metcalf D, Begley CG, Johnson GR, Nicola NA, Lopez AF, Williamsen DJ. Effects of purified bacterially synthesized murine multi-csf (IL-3) on hematopoiesis in normal adult mice. Blood 986;68: I Broxmeyer HE, Williams D, Hangoc G, et al. Synergistic myelopoietic actions in vivo after administration to mice of combinations of purified natural murine colonystimulating factor, recombinant murine interleukin-3, and recombinant murine granulocyte/macrophage colony-stimulating factor. Proc Natl Acad Sci 987;84: Donahue RE, Seehra J, Metzger M, et al. Human IL-3 and GM-CSF act synergistically in stimulating hematopoiesis in primates. Science 988;24: Krumwieh D, Seiler FR. In vivo effects of recombinant colony-stimulating factors on hematopoiesis in cynamolgus monkeys. Transplant Proc 989;2: Mayer P, Valent P, Schmidt G, Liehl E, Bettelheim P. The in vivo effect of recombinant human interleukin-3: demonstration of basophil differentiation factor, histamine-producing activity and priming of GM-CSF-responsive progenitors in nonhuman primates. Blood 989;74: Ulich TR, del Castillo J, Busser K, Guo K, Yin S. Acute in vivo effects of IL-3 alone and in combination with IL-6 on the blood cells of the circulation and bone marrow. Am J Pathol 989; 35: Geissler K, Valent P, Mayer P, et al. Recombinant human interleukin-3 expands the pool of circulating hemopoietic stem cellsin primates-synergism with recombinant human granulocyte/macrophage colony-stimulating factor. Blood 990;75: Ganser A, Lindemann A, Seipelt G, et al. Effect of recombinant human interleukin- 3 in patients with normal hematopoiesis and in patients with bone marrow failure. Blood 990;76: Ganser A, Seipelt G, Lindemann A, et al. Effects of recombinant human interleukin- 3 in patients with myelodysplastic syndromes. Blood 990;76: Ganser A, Lindemann A, Seipelt G, et al. Effects of recombinant human interleukin- 3 in aplastic anemia. Blood 990;76: Gillis G, Urdal DL, Clergenger W, et al. Production of recombinant human colonystimulating factors in yeast. Behring Inst Mitt 988;83: Seipelt G, Ganser A, Link H, et al. Response to interleukin-3 treatment in graft failure after bone marrow transplantation. Cytokines in Hemopoiesis, Oncology and AIDS. Berlin-Heidelberg: Springer-Verlag, 992 (in press). 22 Falk S, Seipelt G, Ganser A, et al. Bone marrow findings after treatment with recombinant human interleukin-3. Am J Clin Pathol 99;95: Ottmann OG, Ganser A, Seipelt G, Eder M, Schulz G, Hoelzer D. Effects of recombinant human interleukin-3 on human hematopoietic progenitor and precur-

12 80 Clinical Trials with Interleukin sor cells in vivo. Blood 990;76: Geissler K, Valent P, Mayer P, et al. Recombinant human interleukin-3 expands the pool of circulating hemopoietic stem cells in primates-synergism with recombinant human granulocyte/macrophage colony-stimulating factor. Blood 99075: Merget RD, Maurer AB, Koch U, et al. Histamine release from basophils after in vivo application of recombinant human interleukin-3 in man. Int Arch All Appl Immunol 990;92: Lindemann A, Henmann F, Mertelsmann R, et al. Human recombinant interleukin- 3: a phase In clinical study. In: Mertelsmann R, Herrmann F, eds. Hematopoietic Growth Factors in Clinical Application. Marcel Dekker Inc, 990: Ganser A, Janssen JWG, Ottmann OG, et al. In vivo effects of granulocyte-macrophage colony-stimulating factor and interleukin-3 on clonal and non-clonal cell populations in patients with clonal hematopoietic disorders. Leukemia 99;5: Ganser A, Lindemann A, Seipelt G, et al. Synergistic effects of sequential IL-3/ GM-CSF treatment in comparison to IL-3 alone in vivo. Blood 990;76(Suppl ): 45a. Hoelzer D, Ganser A, Seipelt G, et al. Hemopoietic growth factors in the treatment of patients with myelodysplastic syndromes. Roc Amer SOC Clin Oncol 990;9:7. Kurzrock R, Talpaz M, Salewski E, et al. Phase I study of recombinant human interleukin-3 in patients with bone marrow failure. J Cancer Res Clin Oncol 990; 6:874. Migliaccio AR, Migliaccio G, Adamson JW. Effect of recombinant hemopoietic growth factors on proliferation of human marrow progenitor cells in serum-deprived liquid culture. Blood 988;72: Messner HA, Yamasadi K, Jamal N, et al. Growth of human hemopoietic colonies in response to recombinant gibbon interleukin-3: comparison with human granulocyte and granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci 987i Oral Discussion Dr. Ihle: My impression from other studies is that one of the most consistent things that one sees with IL-3 in vivo is eosinophilia. And I was wondering, in the last group of patients where you had a variable response with regards to platelet, etc., was there a consistent increase in the eosinophils in those patients in response to IL-3? Dr. Hoelzer: We found in both MDS studies, also with a lower dose, a consistent increase in eosinophils. Dr. McNiece: In the patients that didn t have increased platelet levels, were you able to look at IL-6 levels to see if there was a correlation between increase in platelets with increase in IL-6 production? Dr. Hoelzer: No, we did not. It is clear that IL-6 might mediate the increase in platelet production, but we found no correlation. Of the 4 patients in which we measured IL-6, 6 patients had increased IL-6 levels, but there was no correlation. Questioner: What happens after the withdrawal of the interleukin 3 therapy?

13 Hoelzer/Seipel t/ottmann/ganser 8 Dr. Hoelzer: In most of the patients the neutrophils as well as the platelets drop after four weeks, at the latest after three months, but single patients like the patient I have shown you with long-lasting pancytopenia after intensive treatment (he had multiple myeloma), have persistent increase of platelets. I think in patients where you have sufficient stem cell reserves which are suppressed by chemo- or radiotherapy, you may have a longer lasting effect, but certainly not in patients where the clone is malignant, as for instance, in the myelodysplastic syndromes. Questioner: How about MDS? Dr. Hoelzer: In all of the patients with the MDS the platelet as well as the neutrophils dropped down after weeks or months. Dr. Ogawa: Did IL-3 increase reticulocyte and hemoglobin values in some patients? Dr. Hoelzer: In a substantial proportion of patients we found a reticulocyte increase, however very seldom a corresponding increase in hemoglobin level. Dr. Ogawa: I always thought that there is no effect of IL-3 on erythropoiesis because of the negative feedback regulation by erythropoietin. Is your observation compatible with what Dr. Spivak presented? Dr. Hoelzer: No, it is probably not compatible, but I think the data are consistent insofar as you see an increase in the BFU-e in the peripheral blood, an increase in the proliferation rate of BFU-e and an increase of reticulocytes, the latest state of the erythrocytes in the bone marrow. Dr. Ogawa: Clearly the reticulocytes are under the erythropoietin control, not IL-3. Questioner: What is the longest duration of response to IL-3? Dr. Hoelzer: The longest response I can mention is four years because some of the patients with secondary bone marrow failure or chronic myeloproliferative syndromes who had severe cytopenia after intensive chemotherapy still have normal or subnormal values at that time. I think the mechanism of IL-3 is that it just turns on the early progenitor cells and the cells return to normal or subnormal levels. But as I repeatedly say, in the majority of patients, the values drop down. Dr. Wang: Can interleukin 3 promote leukemic transformation in MDS patients? Dr. Hoelzer: Yes, it can have an effect on the malignant cells, and from the 8 patients with myelodysplastic syndromes we had one patient having a rab-t which means 30% of blood cells. This patient had already a progression before IL-3 treatment and she progressed afterwards to overt leukemia. And I think it is theoretically and in practice it is possible that JL-3 stimulates the malignant cells, especially in acute myeloblastic leukemia. On the other hand, we know from single patients that it stimulates differentiation of the malignant clone also in AML. So in the future we have to find out what the optimal dose is to stimulate the remaining normal clone and not to stimulate the malignant clone. This is the reason why in the last study we used such a low IL-3 dosage for the MDS patients. However, apparently this was too low in this patient because we did not see any increase of blood cells.

14 82 Clinical Trials with Interleukin-3 Questioner: How long did the therapeutic effect last in your patients? Dr. Hoelzer: The therapeutic effect lasted for weeks and occasionally for months. Dr. Schuster: A number of your MDS patients had cytogenetic abnormalities such as the Sq-minus syndrome. After administration of interleukin 3 did these normally-appearing, differentiated cells still have a persistent cytogenetic abnormality? Dr. Hoelzer: Unfortunately, none of our cases had the Sq-minus syndrome. Dr. Seiler: Dr. Hoelzer kept very well to the title of his talk and, therefore, he did not tell us about other studies which were run in his department together with Dr. Mertelsmann s group, and I would like him to comment on the combined use of IL-3 and GM-CSF. Dr. Hoelzer: We did a study priming five days with interleukin 3 followed by ten days of GM-CSF, and in all of the patients there is a more pronounced increase in neutrophils and about the same effect in platelets.