Therapy of advanced acute myeloblastic leukemia with cytarabine and interleukin 2

Leukemia Research Vol. 15, No. 8, pp. 759-763, 1991.

0145-2126/91 $3.00 + .00 Pergamon Press pie

Printed in Great Britain.

CASE REPORT T H E R A P Y OF A D V A N C E D A C U T E MYELOBLASTIC LEUKEMIA WITH C Y T A R A B I N E A N D INTERLEUKIN 2 ANNA BUTTURINI, ENRICHETTABONILAURI,* GIANCARLO IZZI,t GIANFRANCOCROCI,~ FABRIZIAFRANCHI,~M. ALESSANDRASANTUCCI,§M. TERESATONDELLI#and ALESSANDRO CASTRIOTA

SCANDERBEG

Department of Pediatrics, University of Parma; *2nd Clinical Laboratory, USL4, Parma, Italy; tDepartment of Pediatrics, USL4, Parma, Italy; SLaboratory of Cytogenetics, USL9, Reggio Emilia, Italy and §Department of Hematology, University of Bologna, Bologna, Italy (Received 20 January 1990. Revision accepted 7 March 1991) Abstraet--A child with acute myelogenous leukemia who relapsed three months after an allogeneic bone marrow transplant received intermediate-dose cytarabine followed by interleukin 2 (IL-2). Complete remission was achieved after the first cycle of IL-2. Five more combined cycles of cytarabine and IL-2 were given over the next year, during which remission has persisted. IL-2 therapy affected serum tumor necrosis factor (TNF), interferon gamma (IFNy) and soluble IL-2 receptor (slL-2r) levels. In vitro cytotoxicity against leukemia cell lines and recipient leukemia cells was also increased. Key words: Interleukin-2, immunotherapy, acute myelogenous leukemia

that IL-2 treatment can contribute to achieving and maintaining long-term remission in advanced AML.

INTRODUCTION C O N S I D E R A B L E data indicate a role for the immune system in controlling leukemia in humans. Although most prior trials of immune therapy showed no benefit [1], recent analyses of leukemia relapse in recipients of bone marrow transplants suggest that immune-mediated anti-leukemia effects operate. Some of these effects may be independent from graftvs-host disease (GVHD) [2]. Interleukin 2 (IL-2) is a potent immune regulatory molecule which in vitro enhances natural killer (NK) activity and induces a subset of T lymphocytes to kill tumor target cells (lymphokine activated killer-LAK--cells) [3, 4]. Some data suggest that IL-2 might be effective in human leukemias [5-7]. We treated a child with acute myelogenous leukemia (AML) who relapsed shortly after an allogeneic bone marrow transplant with intermediatedose cytarabine followed by IL-2. The child achieved complete remission after the first course of IL-2 and is still in remission after one year. These data suggest

MATERIALS AND METHODS Previous history A two-year-old boy developed AML (FAB type M4) in December 1987. Chromosome analysis of leukemia cells showed a 47, XY, -6, +8, +der(6), t(2;dir dup6) (2qter---> 2q32::6p22--> 6pl 1: :6p22--> 6qter), del(2)(q32), inv(7)(q31q34), del(12)(pl3) karyotype. He achieved remission after one course of cytarabine (100 rag/m/day for 7 days) and daunorubicin (45 mg/m2/day) for 3 days. Post-remission therapy, including intermediate-dose cytarabine (600 mg/m2/day for 5 days), was given until October 1988. In June 1989 leukemia recurred. Multidrug induction therapy, including epidoxorubicin and cytarabine (200 mg/ kg/day), resulted in a second remission. Intensive postremission therapy was given. In October 1989 the child received a bone marrow transplant from his HLA identical sister. Pre-transplant conditioning regimen included cyclophosphamide (120mg/kg) and total body irradiation (12 Gy in 6 fractions). Post-transplant GVHD prophylaxis was cyclosporine A. Engraftment was rapid; modest acute cutaneous GVHD was successfully treated with corticosteroids. Three months after the transplant, the child relapsed with >70% leukemia cells in the blood and bone marrow. An orbital CAT scan, performed because of bilateral

Correspondence to: Anna Butturini, M.D., Dept. of Pediatrics, University of Parma, via Gramsci 14, 43100 Parma, Italy. 759

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exophthalmus, showed infiltration of periorbital tissues presumably'from leukemia. Cytogenetic analysis of the bone marrow showed male cells with identical abnormalities to those detected at diagnosis. Cyclosporine A and corticosteroids were stopped. Treatment

Treatment consisted of cytarabine (500 mg/m 2 every 12 h for 3 days) followed by IL-2 when neutrophils were >0.5 x 109/1. IL-2 (Glaxo IMB, Geneva, CH-specific activity: 1.6 x 106/mg) was given at 200 ~tg/m2/day in 6 h infusion for 5 days. Six courses of cytarabine and IL-2 were given in one year. Hematological and immunological studies

Immune phenotype of blood mononuclear cells was determined by single and double staining with monoclonal antibodies anti-CD2, -CD3, -CD4, -CD8, -CD16, -CD56, -CD25 and anti-DR (Becton Dickson, Mountain View, CA, U.S.A.). Serum levels of soluble IL-2 receptor (slL-2r) and tumor necrosis factor (TNF) were assayed by ELISA (T-Cell Science, Cambridge, MA, U.S.A.); interferon gamma (IFNy) and granulocyte-macrophage colony stimulating factor (GM-CSF) were assayed by RIA (Centocor Inc., Malvern, PA, U.S.A.; Genzyme Corp. Boston, MA, U.S.A.). Detection limits of the commercial assays are 50 U/ml for slL-2r, 10 pg/ml for TNF, 0.1 U/ml for IFNy and 7.5 pg/ml for GM-CSF. Chromosome analyses of PHA stimulated peripheral cells and spontaneously dividing bone marrow cells were performed by conventional techniques. Cytotoxicityof uncultured or IL-2 activated blood mononuclear cells against leukemia cells was assayed as inhibition of leukemia cell colony formation in semi-solid medium [8]. Target cells were K562 and Raji cell lines and recipient leukemia cells, that have been thawed and incubated overnight in RPMI 1640 (Gibco, Grand Island, NY, U.S.A.) supplemented with 10% fetal calf serum (FCS), 1% glutamine, 1% Na pyruvate and antibiotics (complete medium). Effector cells were blood mononuclear cells, separated on Ficoll-Hypaque and resuspended in complete medium. In experiments assessing LAK activity and presence of LAK precursors [9], effector cells were incubated with IL-2 (1000 U/ml) in 5% CO2 at 37°C for 3 h and 5 days respectively, and washed three times before testing. Uncultured and IL-2 activated cells were co-cultivated with target cells at effector : target ratio of 1:1, 10:1 and 50:1 for 18h. Cells were then washed twice and plated in complete medium with 0.8% methylcellulose, 5 × 10-4 mol/l mercaptoethanol and 25% FCS in 24 well plates (Sterilin Limited, Feltham, U.K.). Cultures of recipient leukemia cells were supplemented with 0.1 ttg/ml GM-CSF (Sandoz, Basel, CH). Controls were cultures of effector and target ceils alone. Every assay was done in triplicate. Cultures were incubated at 37°C with 5% CO2 for 5-7 days. Colonies (>40 cells) were counted with an inverted microscope. In no cases did cultures of effector cells alone give rise to colonies. Per cent cytotoxicity was calculated as 100

# colonies grown in presence of effector cells × 100 # colonies grown in absence of effector cells

RESULTS A bone marrow aspirate performed 21 days after the first course of cytarabine showed decreased cellularity with 25% leukemia blasts; cytogenetic studies confirmed the presence of male cells with leukemiaassociated abnormalities. Periorbital infiltration was unchanged. Chromosome analysis of PHA-stimulated blood cells revealed 90% normal male (recipient) metaphases and 10% normal female (donor) metaphases (90 metaphases examined). IL-2 treatment was started 24 days after completing cytarabine treatment, when peripheral neutrophils were 0.5 x 109//1. During IL-2 treatment, neutrophils increased to 6 x 109/1 (Fig. 1 and infra). A bone marrow aspirate performed one week later showed a normocellular marrow, with predominance of mature myeloid cells and increased eosinophils. The exophthalmus resolved completely within one month. Interestingly, PHA-stimulated blood cells now showed only normal female (donor) metaphases (150 metaphases examined). Before IL-2 therapy, uncultured blood cells and blood cells incubated with IL-2 for 3 h had no detectable cytotoxicity to the leukemia cell lines or recipient leukemia cells. However, in vitro antileukemia activity (30-70% cytotoxicity at effector/target ratio 1 : 1) was detected after a 5 day incubation with IL-2. After the fourth day of IL-2 treatment, spontaneous cytotoxicity to cell lines and recipient leukemia cells exceeded 85% and 75% respectively (effector/target ratio = 1 : 1). Levels were not further increased by in vitro incubation with IL-2. Spontaneous antileukemia activity remained high (>50%) between courses, increasing to greater than 90% during the following IL-2 treatments. Each IL-2 treatment resulted in immediate granulocytosis and lymphocytopenia (Fig. 1). After 3-4 days, CD25+, CD56+, C D 1 6 + / - , C D 3 + / - and CD25 +, CD3 + cells were detectable. Lymphocytosis, with presence of cells with activated phenotype, and eosinophilia developed after 5-7 days and persisted for 24-31 days. IFNy serum levels increased during the IL-2 infusions, becoming undetectable by 24 h; TNF levels modestly increased, returning to baseline levels by the end of the courses (Fig. 1). slL-2r levels gradually increased during the five days of treatment, decreasing in 3-7 days, without reaching pre-treatment values. Figure 2 shows the correlation between slL2r and circulating cells expressing membrane IL2r (CD25-positive cells). GM-CSF levels were low (<10 pg/ml), independently from IL-2 treatment or granulocytes in the blood. The child is presently in complete remission for one year. Chromosome analyses of PHA-stimulated

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blood cells and spontaneously dividing bone marrow cells show only normal female (donor) metaphases. The side effects of IL-2 treatment were fever (>40°C) for 1-3 h following every infusion and moderate fluid retention. The child never developed signs of acute or chronic G V H D . DISCUSSION We reviewed features of a child with advanced A M L who appeared to respond to therapy with cytarabine and IL-2. The subject had relapsed twice

previously, the second time only three months after an allogeneic bone marrow transplant. Such persons have an extremely poor prognosis [10]. In our case, duration of the third remission exceeds one year, longer than the previous one. We combined cytarabine and IL-2 because of data suggesting that IL-2 treatment is most effective when tumor mass is relatively small [4]. Because we used cytarabine, we cannot prove that IL-2 was responsible for the results observed. However, some data suggest that in this case IL-2 contributed to achieving and maintaining remission. First, it is unlikely that

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Acknowledgements--Dr Panebianco (Glaxo Spa, Verona, Italy) kindly provided IL-2. This work was supported by the "Noi per Loro" Association on behalf of children with hematological diseases.

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cytarabine alone, at the schedule we used, would control advanced A M L for one year. Also, there was only a partial response after cytarabine, but a complete remission was seen after IL-2. Similar results are reported by others [11]. Finally, in this patient, IL-2 treatment was associated with changes in in vitro tests of anti-leukemia immunity, reinstauration of donor hematopoiesis and secretion of lymphokines with antileukemia activity [12]. All these mechanisms may be responsible for clinical response. The possibility to use immune modulators, such as IL-2, in human leukemias is appealing. Several trials are in progress; preliminary results differ [11, 13, 14]. Many leukemia-, patient- and treatment-related variables could affect the likelihood of cure. For example, leukemia cells might be unresponsive to IL2-mediated effects or might proliferate in response to IL-2 [13] or the other lymphokines induced by IL-2 treatment (Butturini A., Santucci M. A.: unpublished). Also, we observed an increase in serum sIL-2r and cortisol levels in children with advanced malignancies receiving IL-2 [15]. In this patient, IL2-mediated changes in clinical and laboratory parameters were consistent during the different cycles. It is however possible that feed-back mechanisms, generated by IL-2 treatment, could block the antileukemia effects. Large randomized trials are needed to determine the role of IL-2 in treating leukemia.

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Therapy of advanced acute myeloblastic leukemia 13. Macdonald D., Jiang Y. Z. & Swirsky D. et al. (1991) Acute myeloid leukemia relapsing following interleukin-2 treatment expresses the alpha chain of the interleukin-2 receptor. Br. J. Haemat. 77, 43--49. 14. Meloni G., Foa' R. & Vignetti M. et al. (1991) ABMT followed by IL-2 in children with advanced leukemia.

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Abstracts 17th Annual Meeting of EBMT, Cortina d'Ampezzo, Italy 27. 15. Butturini A., Bonilauri E., Terzi C., Izzi C. G. & Giovannelli G. (1990) Effect of IL-2 treatment on immune and hormone levels in children. Blood 76 (suppl. 1), 382a (abstr.).