Phase I Trial of Sequential Topotecan Followed by Etoposide in Adults With

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Leukemia (1999) 13, 343–347  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Phase I trial of sequential topotecan followed by etoposide in adults with myeloid leukemia: a National Cancer Institute of Canada Clinical Trials Group Study M Crump1,3, J Lipton2,3, D Hedley2,3, D Sutton1,3, F Shepherd1,3, M Minden2,3, K Stewart1,3, S Beare4 and E Eisenhauer4

1Department of Medicine, The Toronto Hospital; 2Department of Medicine, Princess Margaret Hospital; 3Faculty of Medicine, University of Toronto, Toronto; and 4National Cancer Institute of Canada Clinical Trials Group, Kingston, Ontario, Canada

Prolonged exposure to a topoisomerase I inhibitor may agent with topoisomerase I inhibition by topotecan produces increase expression of topoisomerase II, making cells more synergistic cell killing in vitro and in vivo.6,7 Sequential susceptible inhibitors of that enzyme. This study was under- taken to establish the maximum tolerated dose (MTD) of a administration of topotecan followed by a topoisomerase II topotecan/topoisomerase II inhibitor sequential combination inhibitor may lead to increased activity of the latter drug that may be active in acute leukemia, and to evaluate the effects through increased expression of topoisomerase II. Hendricks of in vivo exposure to topotecan on topoisomerase II levels in et al8 have shown that resistance to topotecan may be leukemic blast cells as measured by image cytometry. Patients mediated by p-glycoprotein, an important mechanism of drug who were eligible for this phase I study had relapsed or refrac- resistance in AML. However, the effect of p-glycoprotein over- tory acute myeloid leukemia (р2 prior regimens) or CML blast crisis (0 or 1 prior regimen). Topotecan was given as a 5 day expression on topotecan accumulation in cells is substantially continuous i.v. infusion and was to be escalated through three smaller than on the accumulation and cytotoxicity of dauno- levels (1.5, 1.75 and 2.0 mg/m2 day), followed by etoposide at rubicin or VP16 in the same cell line.8 Topotecan remained two dose levels (100 and 150 mg/m2) i.v. bolus days 6, 7 and 8. active in p-glycoprotein expressing tumour xenografts in Topoisomerase II␣ levels in leukemic blasts from bone marrow vivo.9 were measured by image cytometry prior to starting treatment, Two phase I studies of topotecan in acute leukemia, given on day 5 of topotecan infusion and on day 28; and daily during 10,11 topotecan in peripheral blood blasts. Dose-limiting toxicity was as a 5 day continuous infusion, have been reported. These seen in two of six patients at the first dose level (topotecan studies have suggested that the maximum tolerated dose is 1.5 mg/m2/day, etoposide 100 mg/m2/day; уgrade 3 mucositis 2.0–2.3 mg/m2/day for 5 days, and the dose-limiting toxicity in both cases). This cohort was expanded to 10 patients; no is mucositis. No other significant nonhematologic toxicity was further non-hematologic dose-limiting toxicity was observed, observed. Responses were seen in heavily pre-treated patients, but given the extent of toxicity seen, further dose escalation indicating that topotecan may have important activity in leu- was judged not to be feasible. Topo II␣ levels increased in peripheral blood blasts during the first 72 h of topotecan infusion kemia. The primary goal of the present study was to establish and returned to near baseline by day 5, whereas levels the maximum tolerated dose of a topotecan/topoisomerase II appeared to decrease in bone marrow blasts by day 5 com- inhibitor combination which might be incorporated in primary pared to pretreatment. One complete hematologic and cyto- therapy of acute myeloid leukemia. We chose etoposide as genetic remission in a patient with CML blast crisis was the topoisomerase II inhibitor for this study because of its observed in the 10 patients evaluable for response. The demonstrated activity in AML12 and because most patient sequential administration of topotecan 1.5 mg/m2/day continuous infusion for 5 days followed by etoposide treated at our institutions are not routinely exposed to this 100 mg/m2/day × 3 is the recommended phase II dose for this drug during initial therapy. A secondary goal was to evaluate schedule. Topotecan increases topo II␣ expression in vivo in the effect of the administration of topotecan on the expression leukemia cells, but levels of the enzyme are cell cycle depen- of topoisomerase II in leukemic blasts from marrow and dent. Pharmacodynamic evaluation of the sequential or combi- peripheral blood of patients with AML. nation administration of novel antileukemic agents may help improve treatment strategies in acute leukemia. Keywords: topotecan; topoisomerase II; acute leukemia Methods

Introduction Eligibility criteria

Topotecan is a semi-synthetic water soluble analogue of Patients were eligible for this trial if they had relapsed or camptothecin, which inhibits the nuclear enzyme topoisomer- refractory acute myeloid leukemia and had received one or ase I. This novel chemotherapy agent has shown broad two prior regimens. Patients with chronic myeloid leukemia activity in solid tumors, especially carcinomas of the lung and (CML) in blast phase and patients with secondary acute ovary.1 In preclinical studies, topotecan was highly active in myeloid leukemia following a diagnosis of myelodysplasia mice bearing L1210 leukemia.2 Experimental data suggested (MDS) who were previously untreated or had received one that prolonged exposure to topotecan may result in greater prior regimen were also eligible. Those who had previously activity compared to bolus administration.3 received an autologous or allogeneic bone marrow transplant Levels of topoisomerase I may be higher in malignant cells were excluded. Additional eligibility criteria included an and in cells which are actively dividing.4,5 Preclinical data ECOG performance status р2, serum creatinine р1.5 times suggest that the combination of a topoisomerase II active the upper limit of normal, normal serum bilirubin and a left ventricular ejection fraction measured by a MUGA scan у40%. Those with active or uncontrolled infection or with Correspondence: M Crump, Princess Margaret Hospital, Rm 5-108, 610 University Ave, Toronto, Ontario, Canada M56; Fax 416 946 treatment-related acute myeloid leukemia were excluded. 4564 This trial was approved by the human research ethics commit- Received 28 February 1998; accepted 30 October 1998 tees of the University of Toronto, The Toronto Hospital and Topotecan and etoposide in AML: phase I M Crump et al

344 Ј Princess Margaret Hospital, and all patients provided written ary antibody, F(ab )2 anti-rabbit antibody (Cedarlane, Hornby, informed consent. Ontario, Canada). Counterstaining of nuclear DNA was done with 8 ␮mol DAPI (4,6-diamino-2-phenylindole) for 30 min. The slides were then cover-slipped with a glycerol based Treatment plan mountant with 2.3% 1,4-diazabicyclo[2-2-2]octane (DABCO) made up in 90% glycerol and stored in the dark at 4°C. All patients were treated in hospital and chemotherapy was A quantitative immunofluorescence method was used to administered through an indwelling Hickman catheter. Topo- measure nuclear topo II␣ antibody labelling, as previously tecan was administered by 24 h continuous infusion on days described.14 Analysis was performed using an Olympus, Mis- 1–5 (120 h infusion total), followed by etoposide given over sissauga, Ontario, Canada fluoroscope fitted with an intensi- 1 h on days 6, 7 and 8. Because mucositis was anticipated to fied CCD camera interfaced with a SAMBA 4000 image cyto- be the dose-limiting toxicity of this combination, the initial meter. Using 330–385 nm excitation and 420 nm barrier filter topotecan dose was set below the previously established MTD cube, DAPI staining was used to create a nuclear mask. The (2.0 mg/m2/day for 5 days). The starting dose of topotecan was same field was then viewed with 470–490 nm excitation and 1.5 mg/m2/day, and etoposide 100 mg/m2 daily, with the 515 nm barrier filter cube to measure antibody binding. The intention of escalating topotecan to 1.75 mg and 2.0 mg/day, DAPI nuclear mask was then overlaid on this image so that followed by escalation of etoposide through two-dose levels only specific nuclear immunofluorescence was measured.14 to 150 mg/day. Only the nuclei of large blast-like cells were assessed in both Does-limiting toxicity was defined as NCIC-CTG grade у3 bone marrow and peripheral blood. Depending on the cellu- renal, hepatic or gastrointestinal toxicity (the latter lasting 3 larity of the sample, between 200 and 900 cells were meas- or more days); any sign of congestive heart failure or arrhyth- ured for each slide. Results for antibody labelling were mia; у grade 3 central nervous system toxicity or skin rash; expressed as the integrated fluorescence, which corresponds or grade 2 pulmonary toxicity. In this study hematologic dose- to the total amount of enzyme protein in the nucleus. Slides limiting toxicity was defined as pancytopenia with a hypocel- from the same batch of fixed, frozen ALL cells were used as lular marrow (р5% cellular and no marrow blasts) lasting controls and standards for each staining run, and the results more than 6 weeks from the start of treatment. If two or more for patients were expressed as relative values. patients of the first three treated at a given dose level experienced DLT then that level was considered the maximum tolerated dose, and the next lower dose level would be expanded Results by a further three patients. Patients experiencing anti-leukemia effects could be retreated after recovering from a prior course Eleven patients were enrolled on this trial. Their character- if there was no DLT in that patient; a reduction of one dose istics are summarized in Table 1. All of the patients with AML level was required if DLT had occurred in that patient in the had previously received high-dose Ara-C, either as initial first cycle. Dosage calculations were made on the basis of induction therapy or at first relapse. The first patient entered actual height and weight. Supportive care during therapy was at dose level I was diagnosed with CNS leukemia on day 2 according to usual institutional practice, and included red of the topotecan infusion, and required intrathecal chemo- blood cell transfusion for Hb Ͻ80 g/l and platelet transfusion therapy; therefore this patient was inevaluable for toxicity. for counts Ͻ20 × 109/l. One of the next three patients enrolled at the first level experi- Criteria for complete and partial remission were the same enced grade 3 diarrhea and grade 4 stomatitis requiring intra- as those used previously.13 Patients with CML were con- venous narcotics and total parenteral nutrition for 10 days. sidered to have achieved complete remission if their blood Four more patients were then added to the first dose level, counts and bone marrow were normal, even if the Philadel- and a second patient encountered dose-limiting toxicity (grade phia chromosome persisted in cytogenetic analysis. 3 mucositis requiring intravenous narcotics for 7 days). There did not seem to be any clear relationship between the occur- rence of grade 3 gastrointestinal toxicity, and the degree of Topoisomerase measurements pretreatment of the initial seven patients enrolled at the first

Assessment of leukemic blast cell topoisomerase II levels was performed as described previously.14 Briefly, cytospin prep- Table 1 Patient characteristics (n = 11) arations of leukemia cell lines (provided by Dr M Freedman, Toronto) expressing high levels and low levels of topoisomer- Median age (range) 46 (27–67) ase II were used as positive and negative controls. Bone mar- Gender row smears from study patients were made prior to treatment Female 2 Male 9 and on day 5 of the topotecan infusion. Peripheral blood films Performance status (ECOG) were made daily during topotecan infusion from patients in 01 whom circulating blasts were present and were analyzed simi- 18 larly. Slides were air dried, fixed in neutral-buffered formalin 22 for 10 min and stored at −20°C. Leukemia type At the time of processing the slides were pre-treated with AML 6 CML 5 1% 2-mercaptoethanol and 0.1% SDS for 10 min at room Prior therapy temperature, rinsed, permeabilized with cold methanol for Chemotherapy 11 2 min and then incubated in a humid chamber for 3 h with Oral only 2 anti-topo II␣ (P170) antibody (TopoGEN, Columbus, OH, 1 i.v. regimen 5 USA) diluted 1:50. After rinsing, the slides were incubated for 2 i.v. regimens 4 3 h in a dark humid chamber with a 1:150 dilution of second- Topotecan and etoposide in AML: phase I M Crump et al 345 dose level. Furthermore, the toxic effects experienced in the other five patients at this dose level were in keeping with acceptable toxicity for an AML treatment regimen. Therefore, a decision was made to expand the cohort by a further three patients. No further grade 3 non-hematologic toxicity was noted. All patients developed grade 4 pancytopenia with a median time to neutrophil recovery of 26 days (range 21–33+) and platelet recovery of 25 days (range 18–42+). This degree of myelosuppression is comparable to that seen in our centers with other second-line leukemia protocols. After review of the toxicity of this group of 10 evaluable patients, it was felt that enrollment of patients at the next dose level of 1.75 mg/m2 of topotecan and 100 mg/m2 was likely to result in unacceptable gastrointestinal toxicity and further dose escalation could not be justified. Therefore, dose level I was determined to be the recommended dose for further phase II evaluation. Non- hematologic toxicity experienced by all evaluable patients is summarized in Table 2. Of the 10 patients evaluable for response, there was one complete hematologic and cytogenetic remission in a patient with previously untreated CML blast crisis. This remission lasted for 3. months following a second cycle of topotecan and etoposide (with no DLT observed). Peripheral blood blasts disappeared in all patients following completion of protocol therapy, except for the woman with CNS progression; Figure 1 Mean cellular topoisomerase II␣ (integrated however, this was transient. fluorescence) in bone marrow blast cells from nine patients obtained prior to and immediately following 5 days treatment with topotecan by continuous i.v. infusion. Modified from Ref. 14 with permission of Topoisomerase II analysis the publisher.

A total of nine patients had bone marrow samples both prior to treatment and on day 5 of the topotecan infusion available for analysis. One patient demonstrated a large increase in topoisomerase II␣ relative to baseline following topotecan administration, whereas with the remaining eight patients there was either no signiﬁcant change or a decrease in enzyme level (Figure 1). Although most patients showed a marked decrease in the number of circulating leukemia cells during the 5 day topotecan infusion, sufﬁcient cells remained to make sequential measurements on peripheral blood cells daily. Analysis of blasts in the peripheral blood obtained on each day of topotecan infusion showed an approximate three- fold rise in peripheral blood topoisomerase II␣ levels relative to baseline although there was considerable inter-patient variability, as indicated by the wide spread in inter-quartile values shown in Figure 2. Maximum enzyme expression

Table 2 Non-hematologic toxicity (n = 10)

Toxicity Grade

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Lethargy 2 3 1 Diarrhea 4 1 3 Stomatitis 2 3 1 1 Vomiting 1 Rash 3 3 Hepatic-bilirubin 3 2 1 AST 3 1 Figure 2 Effects of topotecan on topoisomerase II␣ levels in per- ALP 6 ipheral blood blasts: results show the median values for nine patients Dyspnea 1 1 expressed as a percentage of their pretreatment levels, with upper and lower quartile range. Modiﬁed from Ref. 14 with permission of the NCIC-CTG criteria. publisher. Topotecan and etoposide in AML: phase I M Crump et al 346 appeared on about day 3, followed by a decrease to near obvious in samples from patients in this study, however. A baseline by day 5. greater proportion of cells in both the blood and bone marrow

are in G0 at the end of the topotecan infusion, compared to pretreatment (data not shown). The number of patients ana- Discussion lyzed in this phase I study is small, however, and further evaluation of this relationship is important. Acute leukemia Only two classes of drugs have demonstrated significant appears to be an ideal model for evaluating the kinetics of activity in AML, and they form the basis of current induction important target enzymes in cancer chemotherapy and their chemotherapy regimens. These are the nucleoside analogues relationship to the cell cycle using image cytometry. such as cytosine arabinoside (Ara-C), which interfere with Although the number of patients treated was small, we did DNA synthesis by inhibition of DNA polymerase; and inhibi- observe one complete hematological and cytogenetic tors of the nuclear enzyme topoisomerase II, such as daunoru- remission in a patient with CML blast crisis. All other patients bicin, idarubicin, mitoxantrone, and etoposide. The newer with leukemic cells in their peripheral blood experienced analogues of daunorubicin have produced only modest clearing of their blast cells, although this was transient and in improvement in remission rate, remission duration and sur- no other patient was even a partial remission observed. This vival.15 Promising results have been obtained by the addition may reflect the fact that the LD90 values for topotecan for of fludarabine to Ara-C, but the use of this agent as part of leukemic cells from patients grown in leukemia colony- remission induction strategies has not yet produced improved forming assays is very similar to the steady-state concen- results over Ara-C alone.16,17 Although post-remission stra- trations of the active drug obtained when the drug is given by tegies such as use of high-dose Ara-C18 or allogeneic or auto- continuous infusion.10 logous bone marrow transplantation19 may improve the long- Given the modest gains achieved in the last two decades term outcome of patients under the age of 50 or 60, not all in the therapy of adult acute myeloid leukemia, evaluation of trials have shown this.20,21 Many patients are not eligible for new cytotoxic agents with novel mechanisms of action these approaches because of age, performance status, lack of remains a priority in this disease. The single agent activity donor, or relapse before bone marrow transplantation. Long- demonstrated in AML, ALL and myelodysplasia suggests that term follow-up data show that the majority of patients with inhibitors of topoisomerase I such as topotecan are likely to acute myeloid leukemia treated with chemotherapy ultimately find a place in the treatment of acute leukemia.24 We are cur- relapse and very few are cured.22 rently evaluating the antileukemic activity and biologic effects Phase I trials have shown that significant escalation of topo- of the combination of topotecan 1.5 mg/m2/day for 5 days tecan above doses that can be tolerated in patients with solid continuous i.v. infusion, followed by etoposide 100 mg/m2/ tumors are feasible in patients with leukemia, and have day × 3 (the MTD from this trial) in a phase II study in relapsed suggested that this drug is active in previously treated and refractory AML in a less heavily pre-treated patient popu- patients.10,11 Previous work by Rowinsky et al11 suggests that lation. Investigation of topotecan in combination with other levels of the target enzyme for topotecan, topoisomerase I, topoisomerase II inhibitors or with Ara-C25 appears warranted. are higher in AML and ALL blast cells than in bone marrow mononuclear cells, although the number of patients studied was small. Expression of the enzyme in AML cells appeared References to be fairly homogeneous in their study, suggesting that topoisomerase I would be an appropriate target for anti-leukemic 1 Dancey J, Eisenhauer EA Current perspectives on camptothecins therapy. Although Rowinsky et al did not find a correlation in cancer treatment. Br J Cancer 1996; 74: 327–338. between the percentage of cells in the S and G2M phases of 2 Johnson RK, Hertzberg, RP, Kingsbury WD et al. Preclinical profile the cell cycle and topoisomerase I content,11 others have of SK&F 104864, a water soluble analogue of camptothecin. Pro- shown that topoisomerase I levels are higher in cycling com- ceedings of the Sixth NCI-EORTC Symposium on New Drugs in pared to non-cycling cells.5 Inhibition of topoisomerase I by Cancer Therapy, 1989; 301. 3 Slichenmyer WJ, Rowinsky EK, Donehower RC, Kaufman SH. The camptothecin analogues is believed to result in a compensa- current status of camptothecin analogues as antitumor agents. J 23 tory increase in topoisomerase II activity. Natl Cancer Inst 1993; 85: 271–291. Our data are the first to demonstrate changes in topoisomer- 4 Giovanella BC, Stehlin JS, Wall ME, Wani MC, Nicholas AW, Liu ase II␣ levels in leukemic blast cells after in vivo exposure to LE, Silber R, Potmesil M. DNA topoisomerase I target chemo- a topoisomerase I inhibitor. A rise in nuclear staining for topo therapy of human cancer xenografts. Science 1989; 246: 1046– II␣ was observed over the initial 2–3 days of the drug infusion, 1048. 5 Hwong C-L, Chen C-Y, Shang H-F. Increased synthesis and degra- but declined toward the end of the 5 days of topotecan. In dation of DNA topoisomerase I during the initial phase of human contrast, after 5 days, bone marrow blasts exhibited levels of T lymphocyte proliferation. J Biol Chem 1993; 268: 18982–18986. expression of topoisomerase II␣ that were lower than or 6 Anzai H, Frost P, Abbruzzesse JL. Synergistic cytotoxicity with 2Ј- unchanged from baseline. Because bone marrow was sampled deoxy-5-azacytidine and topotecan in vitro and in vivo. Cancer only on day 5, it is unclear if the increase in topoisomerase Res 1992; 52: 2180–2185. II␣ levels that was seen in peripheral blood blasts would also 7 Anzai H, Frost S, Abbruzzesse JL. Synergistic cytoxicity with com- bined inhibition of topoisomerase (topo) I and II. Proc Am Soc have been observed if bone marrow had been sampled earlier Cancer Res 1992; 33: 431. in the topotecan infusion period. 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