| | A phase I and pharmacodynamic study of sequential topotecan and etoposide in patients with relapsed or refractory acute myelogenous and lymphoblastic leukemiaReceived 23 January 2002; accepted 30 April 2002. Abstract We designed a pharmacokinetic and pharmacodynamic phase I study of sequential topotecan (2.55–6.3 mg/m2) by 72h infusion followed by five daily doses of etoposide for patients with refractory acute leukemia based upon synergistic anti-tumor activity of topoisomerase I and II inhibitors in vitro. Eight of the 29 patients achieved bone marrow aplasia and two patients achieved clinical remission. Common grade 3–4 toxicities included hepatic and gastrointestinal dysfunction, and correlated with increased steady-state plasma topotecan concentration. The predicted up-regulation of topoisomerase II activity by topoisomerase I inhibition was not observed at this dose and schedule and may provide insight into the modest anti-leukemia activity of the regimen.
1. Introduction  Topotecan, a camptothecin analogue that targets the DNA metabolizing enzyme topoisomerase I, has shown activity in patients with relapsed or refractory leukemia and myelodysplastic syndromes when given alone and in combination with other effective anti-leukemic drugs [1], [2], [3], [4], [5]. Camptothecin analogues exert their anti-tumor activity by binding covalently to the topoisomerase I-DNA cleavable complex, and preventing re-ligation of single strand DNA breaks. In dividing cells, the advancing replication fork converts these topoisomerase I-DNA adducts into double-stranded DNA breaks leading to replication arrest and cell death. In some studies, a higher intrinsic content of topoisomerase I in tumor cells has been shown to correlate with enhanced sensitivity to this class of agents [6], [7], [8]. Improvements in treatments for acute leukemia likely will require addition to effective cytotoxic agents given in combinations and schedules designed to modulate DNA repair mechanisms and to overcome intrinsic drug resistance. In the pre-clinical models, sequential administration of topoisomerase I and II inhibitors provides additive or synergistic cytotoxicity, possibly due to up-regulation of topoisomerase II activity [9], [10], [11], [12], [13]. Etoposide, a topoisomerase II inhibitor, administered as a single agent has modest antileukemia activity [14]. While anti-tumor activity has been observed, the sequential administration of camptothecin analogues and etoposide to patients with non-hematologic malignancies has been limited by severe myelosuppression despite only modest extra-medullary toxicity [15], [16], [17]. Therefore, the combination of topotecan and etoposide provides a selective, and therefore, favorable toxicity profile as a potential treatment for acute leukemia where bone marrow aplasia is generally required in order to achieve remission. We designed a phase I study of escalating doses of topotecan followed by a fixed dose of etoposide for patients with relapsed/refractory acute leukemia. Steady-state plasma concentrations of total topotecan and the active lactone form were correlated with response and toxicity. Companion laboratory studies were designed in order to determine whether pre-treatment of topoisomerase I and II activity and modulation of enzyme activity in leukemic blasts during treatment correlated with clinical response. 2. Patients and methods 2.1. Patients Adult patients (≥18 years) with relapsed, refractory acute myelogenous (AML) or acute lymphoblastic leukemia (ALL), acute leukemia secondary to pre-existing hematologic disorder or chemotherapy, or chronic myelogenous leukemia (CML) in blast crisis were eligible for this study. In general, patients believed to be candidates for curative therapy with autologous or allogeneic stem cell transplantation were not enrolled. Adequate cardiac, pulmonary, hepatic and renal function (defined as creatinine clearance ≥60ml/min, total serum bilirubin ≤2.0mg/dl and transaminases ≤3× the upper limit of normal), ECOG performance status ≤3, and a life expectancy of at least 1 month without definitive treatment were required. Prior chemotherapy including etoposide was allowed, and a minimum of 2 weeks must have elapsed since last cytotoxic therapy (excluding hydroxyurea). Exclusion criteria included previous treatment with camptothecin analogues, allogeneic or autologous stem cell transplants, uncontrolled infections, or CNS leukemia. The clinical protocol was approved by the Institutional Review Board at University Hospitals, Case Western Reserve University and all patients gave written informed consent. 2.2. Treatment plan and topotecan dose escalation schema Topotecan (supplied by the Cancer Therapeutics Evaluation Program, National Cancer Institute, Bethesda, MD) was administered as a 72h continuous infusion at a starting dose of 2.55mg/m2 over 72h and escalated by 0.75mg/m2 in cohorts of the three patients (Table 3). Topotecan was diluted to a final concentration of 10–500μg/ml in 0.9% sodium chloride solution and fresh reconstituted solution was mixed and administered every 24h. The starting dose of topotecan was based upon the maximum tolerated dose (MTD) designed by Eckhardt et al. in a phase I study for solid-tumor patients [18]. Etoposide, at a fixed dose of 100mg/m2 was administered as a 1h four infusion for 5 consecutive days, beginning 24h after the completion of topotecan. Chemotherapy was given according to the lesser of actual or corrected ideal body weight. A diagnostic bone marrow aspirate and biopsy was obtained 7 days after the completion of etoposide (days 16–18 from beginning of treatment). Patients with residual leukemia on bone marrow biopsy at this evaluation were eligible for a second induction cycle to begin no sooner than day 16 and no later than day 35 after the beginning of induction cycle 1. Treatment with hematopoietic growth factors was initiated in patients who achieved bone marrow aplasia or <5% blasts. Patients who achieved a complete or partial response could receive one cycle of consolidation therapy with sequential topotecan and etoposide with dose modifications according to toxicity encountered during their initial induction therapy. No dose escalations were allowed within individual patients. Toxicities were graded by the NCI common toxicity criteria (original version). Dose-limiting toxicity (DLT) was defined as a grade 3 or worse non-hematologic toxicity. Grade 3 or greater stomatitis, diarrhea or infectious complications were not considered dose-limiting since these are commonly observed during leukemia induction therapy. Dose-limiting hepatotoxicity was defined as greater than equal to grade 4 hyperbilirubinemia or greater than grade 3 transaminase elevation which did not resolve to <3× the upper limit of normal by day 35 of treatment. Dose-limiting hematologic toxicity was defined as an absolute neutrophil count ≤200per μl or an unsupported platelet count ≤20,000per μl or a ≤5% cellular bone marrow without evidence of residual leukemia lasting more than 4 weeks from the beginning of the most recent cycle of chemotherapy. Deaths occurring within 6 weeks of the beginning of protocol treatment were classified into three categories: (1) disease related, for example, due to leukostasis, (2) cytopenia-related (providing cytopenia was <4 week’s duration), such as infection or bleeding, (3) treatment-related, for example, due to organ toxicities, such as mucositis, hepatotoxicity, or unexpected toxicities not related to bleeding and infection. The dose escalation schema was modified for category 3 deaths only. If no patient at an individual dose level developed a DLT, dose escalation proceeded. If one out of the three patients developed a DLT as defined above, three additional patients were enrolled at the same dose level. If less than two out of the six patients develop DLT dose escalation continued. Each individual in a cohort was followed for a minimum of 6 weeks before the next cohort was entered. Additional patients were enrolled at the current dose level to replace patients who died from categories 1 and 2 causes within 6 weeks from the start of treatment. The dose level below which two or more dose-limiting toxicities were observed was defined as the MTD. 2.3. Laboratory studies 2.3.2. Topotecan total and lactone levels Based on the pharmacokinetics of our previous phase I trials as well as worked by others, plasma lactone and total topotecan concentrations approach steady-state after 10h and are maintained throughout a 72h topotecan infusion [23], [24], [25]. Therefore, a single sample of blood obtained by venipuncture 68h after the start of the topotecan infusion and while the topotecan solution was infusing was chosen to determine steady-state plasma total and lactone topotecan concentration. The 3ml of the whole blood was collected in a heparinized syringe, transferred to a 5ml red-top tube and centrifuged at 4°C for 5min at 3000rpm and transferred into a glass tube. Exactly 1ml of plasma was immediately pipetted to a borosilicate glass tube containing 5ml of methanol (from −20°C freezer), vortex mixed for 10s, and spun in 4°C centrifuge at 3000rpm for 5min. The resulting supernatant was stored at −70°C until analysis. Topotecan lactone and total topotecan were determined using high-pressure liquid chromatography (model 1050 pump, 1046A fluorescence detector, Hewlett-Packard) with minor modifications of sample quantify and reconstitution and volumes of previously published procedures [26]. Quality control samples of high and low concentrations plasma total topotecan and topotecan lactone were determined with each set of patient samples. The control limits (mean±S.D.) for the high and low total topotecan concentration were 7.4±0.7 and 1.1±0.1ng/ml, respectively. For topotecan lactone the control limits were 5.8±0.8 and 1.0±0.2ng/ml for the high and low concentrations, respectively. 2.4. Clinical evaluation Complete response was defined as neutrophil count ≥1200per μl, platelet count ≥100,000per μl without circulating leukemic blasts and bone marrow cellularity of ≥20% with ≤5% blasts. A partial response included all criteria listed previously except that the bone marrow contained 5–25% blasts. Other clinical responses of interest included clearing of peripheral blasts and reduction of bone marrow cellularity on days 16–18 of treatment to <10% and/or blast percentage to <5% (defined as bone marrow response). 2.5. Statistical analysis Unless otherwise noted, data are presented as median and range. The Wilcoxon signed rank test was used to determine correlations between topotecan concentration and toxicity or response. Wilcoxon rank sum was also used to correlate pre-treatment topoisomerase I and II activity and changes in enzyme activity during treatment with clinical response in bone marrow and the peripheral blood. The relationship between down-regulation of topoisomerase I activity during treatment and modulation of topoisomerase II was explored using linear regression analysis. The χ2-test was used for all other statistical evaluations. 3. Results 3.1. Patient characteristics Patient characteristics are listed in Table 1. A total of 29 adult patients were enrolled on this trial between August 1994 and November 1999 and included 22 with AML, six with ALL, and one with CML in blast crisis. Median age was 63 years with a range from 27 to 75 years. One patient with AML secondary to myelodysplastic syndrome was enrolled and treated on protocol after developing recurrent panycytopenia and cytogenetic abnormalities following standard induction chemotherapy. In retrospect, his bone marrow at the time of treatment was noted to have fewer than the required 5% bone marrow blasts, rendering him ineligible for protocol. He was evaluable for toxicity, but no response. Two patients, one with CML in blast crisis and one with AML secondary to prolonged treatment with carboplatin, had not previously received intensive induction treatments. Seven patients (six with AML, one with ALL) were treated in first relapse and had a median duration of first remission of 15 months (range 2–19 months). At the time of protocol treatment, 18 patients had either primary refractory leukemia or had been refractory to standard salvage chemotherapy. A total of 18 of the 29 patients had achieved a complete remission (median duration of 6.5 months, range 1–20) at some time since diagnosis with standard induction therapies. Nine patients had pre-existing myelodysplastic syndromes. 3.2. Clinical responses Two patients, one with acute myelogenous leukemia in first relapse treated at dose level 2 and one with ALL in the first relapse treated at dose level 4 achieved a complete remission of >60 and 2 months, respectively. Clearing of peripheral blood blasts was observed in 11 of the 18 patients and occurred within 6 days of initiating therapy. A total of 27 of the 29 patients underwent diagnostic BM biopsy on days 16–18 from the start of treatment. Total 10 patients, including the two who attained complete remission, achieved a bone marrow response (defined as a reduction of bone marrow cellularity on days 16–18 of treatment <10% and/or blast percentage to <5%). Six of these patients were treated in the first relapse and four patients had primary refractory or refractory/relapsed acute leukemia. Despite achieving bone marrow aplasia, a meaningful improvement in blood counts was not observed in the eight patients who did not achieve clinical remission. Two patients treated in the first relapse and one patient with primary refractory AML who failed protocol therapy subsequently achieved clinical remission after receiving alternate salvage chemotherapy. There did not appear to be any relationship between dose level of topotecan and clinical response. Pre-treatment patient characteristics that were evaluated for potential association with tumor response included treatment in first relapse versus treatment for refractory disease or other subsequent relapse, any previous remission versus no remission, and pre-existing myelodysplastic syndrome (Table 2). Six of the seven patients treated in the first relapse achieved a bone marrow response compared to four of the 18 who were primary refractory or in later relapse (P=0.014, χ2-test). Patients with pre-existing myelodysplastic syndromes and patients who ever achieved a complete remission were not more likely to achieve a bone marrow response than those who did not share these characteristics. 3.3. Toxicity evaluation A total of 20 patients received one cycle of sequential topotecan and etoposide and nine patients received two cycles as either second induction (7 points) or consolidation (2 points). Although dose-limiting toxicity as defined above were not observed at the final dose of 6.3mg/m2 of topotecan given over 72h, the protocol was terminated due to slow patient accrual and minimal efficacy of the regimen. Three deaths occurred, all in patients over 60 years old. Two deaths occurred at dose levels 2 and 3 on days 6 and 20 of the treatment, and were due to progressive pneumonia and poly-microbial bacteremia, without concurrent significant mucositis or diarrhea. The third death was treatment-related due to the development of intractable grade 4 hemorrhagic diarrhea (see Section 2). As anticipated, all patients developed grades 3 and 4 hematologic toxicities and all except two patients developed grade 3 infections. Other non-hematologic toxicities of grade 3 or greater are shown in Table 3. Reversible grade 3 or 4 hyperbilirubinemia, mucositis, and diarrhea were commonly observed at all dose levels. Dose-limiting hepatotoxicity was originally defined as development of grade 4 or greater hyperbilirubinemia or grade 3 or greater transaminase elevation. After three of the six patients entered the dose level 2 developed dose-limiting reversible hepatotoxicity as defined above without apparent serious sequela, CTEP requested treatment of an additional three evaluable patients at a dose level 2 before allowing further dose escalation. Subsequently, a protocol amendment allowed dose escalation, providing hepatotoxicity resolved to <3× the upper limit of normal by day 35 of the treatment. Median duration of grade 3 or greater mucositis was 5 days (range 1–11) and median duration of grade 3 or greater diarrhea was 3 days (range 1–22). Unexpected toxicities included one case of reversible sensorimotor neuropathy and another of symptomatic bradycardia in a patient subsequently found to have coronary artery disease. Two patients who developed grade 4 hyperbilirubinemia during cycle 1 were able to tolerate cycle 2 with a 25% dose reduction. Five patients received a second cycle of induction chemotherapy at full dose without developing at any of the grades 3/4 non-hematologic toxicities.  | Topo dosea (mg/(m272h)) | 2.55 (1) | 3.30 (2) | 4.05 (3) | 4.80 (4) | 5.55 (5) | 6.3 (6) | |
| Bilirubin | 2 (3, 4)b | 2 (3, 4) | 1 (3) | 1 (3) | 3 (3, 4) | 1 (3) | |
| Cardiac | 0 | 1 (3)c | 1 (4)d | 0 | 0 | 0 | |
| Death | 0 | 1e | 1e | 0f | 1d | 0 | |
| Diarrhea | 0 | 1 (3) | 1 (3) | 2 (3, 4) | 2 (3, 5) | 0 | |
| Hemorrhage | 0 | 1 (3) | 0 | 0 | 1 (5) | 0 | |
| Mucositis | 2 (4, 4) | 1 (4) | 0 | 1 (3) | 4 (3, 4) | 1 (3) | |
| Neuro | 0 | 1 (3, 4)g, 0 | 0 | 0 | 0 | 0 | |
| Transaminase | 0 | 1 (3) | 0 | 0 | 0 | 0 | |
| Number of points/number of cycles | 3/5 | 11/13h | 4/5 | 3/4 | 6/6 | 2/3 | | | | |
|
a
Dose levels of topotecan are indicated in parenthesis.
b
Numbers in parentheses indicate grade of toxicity.
c
Atrial fibrillation in patient with severe pneumonia.
d
Bradycardia and chest pain in patient subsequently found to have coronary artery disease.
e
Infectious cause.
f
Death due to grade 4 treatment-related hemorrhagic diarrhea.
g
Reversible sensory motor neuropathy, homonymous hemianopsia due to CNS bleed.
h
Nine evaluable patients: one patient refused further treatment after fourth dose of etoposide, one early death due to infection. |
The time to hematopoietic recovery could be determined for the two patients who achieved a complete remission and one patient with myelodysplastic syndrome and included three courses of induction and two courses of consolidation. Neutrophil recovery (defined as an absolute neutrophil count of 500 per μl) occurred 22, 19, and 29 days from the start of the most recent cycle induction and 20 and 22 days from the start of consolidation. Platelet recovery (defined as the first day of platelet transfusion independence) occurred 30, 21, 20 days from the start of induction chemotherapy and days 22 and 25 from the beginning of consolidation. 3.5. Pharmacodynamic evaluation Topoisomerase I and II activity was measured from the bone marrow aspirates before treatment and 24h after the completion of the topotecan infusion, before beginning etoposide. Pre-treatment topoisomerase I and II activity could be assessed in 25 patients. In contrast to expected results, topoisomerase II activity increased in only 3 patients, each of whom had AML (Fig. 3A). Moreover, there was a significant correlation between the decreases in topoisomerase I and II activities (Fig. 3B, P=0.009, linear regression analysis). We evaluated whether a significant depletion of topoisomerase I may be necessary in order to up-regulate topoisomerase II enzyme activity. In fact, none of the five patients who had had >70% depletion of pre-treatment topoisomerase I activity were observed to have up-regulation of topoisomerase II activity. In order to determine whether up-regulation of topoisomerase II activity may occur earlier during the topotecan infusion, serial blood samples were analyzed for topoisomerase II activity in patients with adequate numbers of circulating blasts. Only one of these four patients had increased topoisomerase II activity during the first 48h of treatment (Fig. 4). In contrast to the expected findings, patients with bone marrow response were not more likely to have higher topoisomerase I and II activity in bone marrow aspirates before treatment compared to those who did not. Furthermore, up-regulation of topoisomerase II activity in the bone marrow or peripheral blood in the three patients mentioned previously was not associated with bone marrow response. On the other hand, there was a higher chance of bone marrow response in the patients who had >70% decrease in topoisomerase I activity compared to those who had a more modest decline in topoisomerase I activity (4/6 versus 2/16, P=0.045). 4. Discussion We designed a phase I and translational study of sequential topotecan and etoposide in patients with relapsed or refractory acute leukemia. We found that plasma concentration, but not dose, of topotecan significantly correlated with treatment-related toxicity. Serial analyses of bone marrow samples of topoisomerase I and II activity in the patients undergoing treatment were done to elucidate pharmacodynamic interactions between topoisomerase inhibitors which may predict tumor response. Sequential topotecan and etoposide was well-tolerated in these extensively treated patients. The observed treatment-related mortality of 10% is comparable to other standard induction regimens. The most commonly observed grade 3–4 non-hematologic toxicities included mucositis, diarrhea, and reversible hepatic dysfunction. At the highest dose level studied of 6.3mg/m2 of topotecan given over 72h followed by 5 consecutive days of etoposide, we have not observed dose-limiting toxicities. Two other phase I leukemia studies have established the MTD of topotecan given as a 5-day infusion in combination with etoposide in the range of 5–7.5mg/m2, with mucositis as the pre-dominant toxicity [4], [27]. In the three patients who recovered hematopoiesis, the duration of marrow aplasia was comparable to that observed with standard dose induction therapy followed by hematopoietic growth factor support [28], [29]. In our study, two of the seven patients treated in first relapse attained a second complete clinical remission of 2 and >60 months duration. No clinical responses were observed among patients with refractory or multiply relapsed acute leukemia. In other studies, sequential topotecan and etoposide has shown similarly low response rates in heavily pre-treated leukemia patients [4], [27]. Interestingly, three of our patients who failed protocol therapy were able to achieve clinical remissions with subsequent treatment. At the dose range studied, we found significant inter-patient variation of steady-state lactone and total topotecan plasma concentrations within dose levels and did not observe a significant relationship between dose and plasma concentration of topotecan. These data may explain, in part, why increased toxicity or anti-leukemia activity was not observed at higher dose levels. The consistent ratio of steady-state lactone to total topotecan plasma levels suggests that sample processing and analysis were accurate. Moreover, increased plasma concentrations of topotecan were associated significantly with the development of grade 3–4 non-hematologic toxicities. Although some investigators have found a linear relationship between topotecan dosage and plasma concentrations at similar dose ranges to our study [30], [31], other investigators have not observed dose dependent pharmacokinetics [25], [32]. Furthermore, in phase II studies up to a 10-fold inter-patient variability between dose administered and topotecan pharmacokinetics has been observed [24], [33], [34]. Moreover, other investigators observed that topotecan plasma concentrations rather than dose administered correlate with tumor response and the development of hematologic and gastrointestinal toxicities [24], [25], [32], [35]. Gallo et al. have proposed a population-based pharmacokinetic model for topotecan dosing which incorporates measures of patient size and renal function [36]. These data suggest that future studies may warrant consideration of dosing based on area under the curve (AUC) [33], [35] or concentration at steady state (present report, [23], [24]). Although pre-clinical studies have shown that increased topoisomerase I activity in tumor cells before treatment may predict cytotoxicity of topoisomerase I inhibitors, this finding has not consistently been demonstrated in acute leukemia, either in vitro or in the clinical setting [1], [37], [38]. Pre-treatment topoisomerase I activity assays were measured in 26/29 of our patients and in this relatively small number of heavily pre-treated patients, increased topoisomerase I levels before treatment did not appear to be associated with achieving either a clinical or bone marrow response. In addition to reduced topoisomerase I content, many other mechanisms of resistance to topoisomerase I inhibitors are likely to be important [6], [7], [8], [39], [40], [41]. These include mutations of the topoisomerase I enzyme with resultant diminished formation of topoisomerase I DNA adducts and decreased sensitivity to topoisomerase I inhibitors, as well as increased ability to repair double-stranded DNA damage [42], [43], [44], [45], [46], [47]. On the other hand, it is possible that modulation of topoisomerase activity may predict response. In our study, a 70% or greater decrease in topoisomerase I activity during topotecan treatment tended to be associated with bone marrow response. Evaluation of peripheral blood or bone marrow for cell cycle distribution and apoptotic fractions may be instructive to further evaluate the relationship between modulation of topoisomerase I activity and response. Many pre-clinical studies, particularly in solid-tumor cell lines and xenograft systems, have shown that sequential administration of topoisomerase I and II inhibitors provides synergistic anti-tumor activity [9], [10], [11], [12], [13], [48]. One potential mechanism for this observation is that deficiency of one topoisomerase enzyme leads to compensatory increases in the alternate topoisomerase enzyme and thereby augmented sensitivity to inhibition of the alternate enzyme. In contrast to our expectations, we observed down-regulation of topoisomerase II activity in the bone marrow aspirates in the majority of patients after the completion of topotecan treatment. In fact, a dramatic decline in topoisomerase I and II activity was observed in both patients who achieved complete remission and no responses occurred in patients in whom up-regulation of topoisomerase II was observed. Because our studies were obtained on mononuclear cell fractions of the bone marrow aspirates, changes in enzyme activity levels observed could, in part, be influenced by variation in blast cell content of the bone marrow during treatment. Our results are consistent with a number of other clinical trials in which pharmacodynamic changes in topoisomerase activity have been explored in serial blood, marrow, and tumor samples from patients receiving topotecan–etoposide combinations [4], [15], [49]. Nair et al. evaluated topoisomerase IIα mRNA and protein levels from peripheral blood leukocytes and lymphoma cells in patients with lymphoma and leukemia and observed consistent decline in topoisomerase II levels after topotecan therapy [49]. Crump et al. also observed down-regulation of topoisomerase II in leukemia patients after topotecan treatment using an immunofluorescent antibody technique allowing quantitation of topoisomerase II levels specifically in nuclei of blast-like cells [4]. It appears that modulation of topoisomerase II by topoisomerase I inhibitors and resultant tumor cytotoxicity is complex and may depend on many factors including tumor cell type, the proportion of cells undergoing nucleic acid synthesis, and induction of p53 [30], [48], [49], [50], [51], [52]. Following treatment with topotecan, Whitacre et al. observed decreased topoisomerase IIá levels in all the three lymphoid cell lines and variable changes in topoisomerase IIá levels in breast and colon cell lines when grown as xenografts in nude mice [50]. Stahl et al. found that incubation of lung and gastric cell lines with camptothecin resulted in a significant down-regulation of topoisomerase II protein expression [53]. Interestingly, despite this observation, additive cytotoxic activity of camptothecin and etoposide was observed. Nair et al. found that DNA damaging agents such as topotecan may up-regulate p53 with resultant down-regulation of topoisomerase IIα gene transcription in leukemia cell lines [49]. Other groups have found subadditive or antiagonistic anti-tumor activity of topotecan combinations which is potentially explained by decreased cellular proliferation and up-regulation of p53 [11], [48], [49], [54]. Kaufman [54] observed antagonism between camptothecin and topoisomerase II-directed chemotherapeutic agents given concurrently to HL-60 cell lines. Further experiments revealed that camptothecin treatment inhibited nucleic acid synthesis, which was required for etoposide cytotoxicity [54]. In a tumor xenograft model, Kim et al. found potentiated adriamycin cytotoxicity in two of the six cell lines in which CPT-11 pre-treatment increased S phase population and topoisomerase II mRNA. In contrast, antagonism between the two agents was found in the cell lines in which these effects did not occur [11]. Therefore, evaluation of cell cycle distribution and p53 expression, in addition to modulation of topoisomerase II activity in the bone marrow aspirates may be of value in understanding and predicting clinical activity of the topotecan–etoposide combination in future studies. Although patients with acute leukemia that develop recurrence within 1 year of achieving complete remission rarely achieve meaningful clinical remissions, the appropriate time to use investigational strategies in these patients and other high-risk patients remains controversial [55], [56]. In our study, patients who were treated in the first relapse were more likely to attain a second complete remission or bone marrow response than those in later stages of their illness. If residual leukemia was found at days 16–18 of the therapy, patients were allowed to receive alternative salvage treatments. Interestingly, two patients treated in first relapse subsequently achieved a complete remission with standard salvage treatments. Thus, it did not appear that exposure to investigational treatments first had an adverse effect on survival and in fact, may allow for a more objective evaluation of promising new treatments. In this phase I study, we found that sequential topotecan and etoposide although well-tolerated, had minimal anti-leukemia activity in heavily pre-treated patients. Furthermore, our pharmacokinetic studies showed significant inter-patient variability in the plasma concentrations of topotecan and the active lactone form, suggesting that doses should be individualized. In correlative laboratory studies, we did not observe the expected up-regulation of topoisomerase II at the dose and schedule of topotecan studied. These data may provide an explanation why sequential topotecan and etoposide treatment did not have additive or synergistic anti-leukemia activity in these high-risk patients. Our study shows that pharmacokinetic studies of chemotherapeutic agents as well as pharmacodynamic evaluation of tumor cells is feasible and that information obtained from patients during therapy may be important in predicting both toxicity and efficacy of novel regimens and in designing new treatment strategies. Acknowledgements Supported, in part, by the Cancer Therapeutics Evaluation Program, Ireland Cancer Center, and NIH Grants U01A62502-07 and M01 RR0080-38. B.W. Cooper provided the concept, design, analysis of the data, drafted and revised the manuscript. E. Donaher collected and assembled the data. H.M. Lazarus provided study materials and critical revision of the paper. S.B. Green gave statistical expertise. D. Gosky, N.S. Rosenthal, S.J. Berger, X. Li and S.T. Ingalls provided technical and/or logistical support. C.L. Hoppel provided technical support and contributed to the revision. S.L. Gerson provided study material and helped with the revision. All authors gave approval to the manuscript. References [1].
[1]
Rowinsky EK, Adjei A, Donehower RC, Gore SD, Jones RJ, Burke PJ, et al.
Phase I and pharmacodynamic study of the topoisomerase I-inhibitor topotecan in patients with refractory acute leukemia.
J. Clin. Oncol. 1994;2:2193–2203. [2].
[2]
Kantarjian HM, Beran M, Ellis A, et al.
Phase I study of topotecan, a new topoisomerase I inhibitor in patients with refractory or relapsed acute leukemia.
Blood. 1993;81:1146–1151. MEDLINE [3].
[3]
Beran M, Kantarjian H, Keating M, et al.
Results of topotecan and high dose cytosine arabinoside (ara-C) in previously untreated patients with high-risk myelodysplastic syndrome and chronic myelomonocytic leukemia.
Blood. 1997;90:2593a. [4].
[4]
Crump M, Lipton J, Hedley D, Sutton D, Shepherd F, Minden M, et al.
Phase I trial of sequential topotecan followed by etoposide in adults with myeloid leukemia. A National Cancer Institute of Canada Clinical Trials Group Study.
Leukemia. 1999;13:343–347. MEDLINE [5].
[5]
Seiter K, Feldman EJ, Halick D, Traganos F, Darzynkiewicz A, Lake D, et al.
Phase I clinical and laboratory evaluation of topotecan and cytarabine in patients with acute leukemia.
J. Clin. Oncol. 1997;15:44–51. [6].
[6]
Hertzberg RP, Carang MJ, Hecht SM.
On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme DNA complex.
Biochemistry. 1989;28:4629–4638. [7].
[7]
Bjornsti MA, Benedetti P, Viglianti GA.
Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the anti-tumor drug camptothecin.
Cancer Res. 1989;49:6318–6323. MEDLINE [8].
[8]
Madden KR, Champoux JJ.
Overexpression of human topoisomerase I in baby hamster kidney cells: hypersensitivity of clonal isolates to camptothecin.
Cancer Res. 1992;52:525–532. MEDLINE [9].
[9]
Kano Y, Suzuki K, Akutsu M, Suda K, Inoue Y, Yoshida M, et al.
Effects of CPT-11 in combination with other anti-cancer agents in culture.
Int. J. Cancer. 1992;50:604–610. MEDLINE |
CrossRef
[10].
[10]
Bertrand R, O’Connor PM, Kerrigan D, Pommier Y.
Sequential administration of camptothecin and etoposide circumvents the antagonistic cytotoxicity of simultaneous drug administration in slowly growing human colon carcinoma HT-29 cells.
Eur. J. Cancer. 1992;28A:743–748. MEDLINE [11].
[11]
Kim R, Hirabayashi N, Nishiyama M, Jinushi K, Toge T, Okada K.
Experimental studies on biochemical modulation targeting topoisomerase I and II in human tumor xenografts in nude mice.
Int. J. Cancer. 1992;50:760–766. MEDLINE |
CrossRef
[12].
[12]
Eder JP, Chan V, Wong J, Wong YW, Ara G, Northey D, et al.
Sequence effect of irinotecan (CPT-11) and topoisomerase II inhibitors in vivo.
Cancer Chemother. Pharmacol. 1998;42:327–335. MEDLINE |
CrossRef
[13].
[13]
Whitacre CM, Zborowska E, Gordon NH, Mackay W, Berger NA.
Topotecan increases topoisomerase IIα levels and sensitivity to treatment with etoposide in schedule dependent process.
Cancer Res. 1997;57:1425–1428. MEDLINE [14].
[14]
Bishop JF.
Etoposide in the treatment of leukemia.
Semin. Oncol. 1992;19(Suppl 13):33–38. Abstract | Full Text |
Full-Text PDF (116 KB)
[15].
[15]
Hammond LA, Eckardt JR, Gamapaathi R, Burris HA, Rodriguez GA, Eckhardt SG, et al.
A phase I and translational study of sequential administration of the topoisomerase I and II inhibitors topotecan and etoposide.
Clin. Cancer Res. 1998;4:1459–1467. MEDLINE [16].
[16]
Ando N, Eguch K, Shinkai I, Tamura T, Ohe Y, Yamamoto N, et al.
Phase I study of sequentially administered topoisomerase I inhibitor (irinotecan) and topoisomerase II inhibitor (etoposide) for metastatic non-small-cell lung cancer.
Br. J. Cancer. 1997;76:1494–1499. MEDLINE [17].
[17]
Herben VMM, Bokkel Huinink WW, Dubbelman AC, Mandjes IAM, Groot Y, Gortel-van Zomeran DM, et al.
Phase I and pharmacological study of sequential intravenous topotecan and oral etoposide.
Br. J. Cancer. 1997;76:1500–1508. MEDLINE [18].
[18]
Eckhardt JR, Burris HA, Rodriguez GA, Fields SM, Rothenberg ML, Moore TD, et al.
A phase I study of the topoisomerase I and II inhibitors topotecan and etoposide.
Proc. ASCO. 1993;12:349a. [19].
[19]
Boyum A.
Separation of leukocytes from blood and bone marrow.
Scand. J. Clin. Lab. Invest. 1968;21:97.
CrossRef
[20].
[20]
Willson J, Gerson S, Haaga J, Berger S, Berger N.
Biochemical modulation of drug resistance in colon cancers.
Proc. AACR. 1992;33:236. [21].
[21]
Duget M, Lavenot C, Harper F, et al.
DNA topoisomerases from rat liver: physiologic variation.
Nucleic Acid Res. 1983;11:1059–1075. MEDLINE [22].
[22]
Champoux JJ, McConaughy B.
Purification and characterization of the DNA untwisting enzyme for rat liver.
Biochemistry. 1976;15:4638–4642. [23].
[23]
Sabiers JH, Berger NA, Berger SJ, Haaga JR, Hoppel CL, Willson JKV.
Phase I trial of topotecan administered as a 72 h infusion.
Proc. AACR. 1993;34:426. [24].
[24]
Rose PG, Gordon NH, Fusco N, Fluellen L, Rodriguea M, Ingalls ST, et al.
A phase II and pharmacokinetic study of weekly 72 h topotecan infusion in patients with platinum-resistant ovarian carcinoma.
Gynecol. Oncol. 2000;78:228–234. MEDLINE |
CrossRef
[25].
[25]
Stewart CF, Baker SD, Heideman RL, Jones D, Crom WR, Pratt CB.
Clinical pharmacodynamics of continuous infusion topotecan in children: systemic exposure predicts hematologic toxicity.
J. Clin. Oncol. 1994;12:1946–1954. [26].
[26]
Beijnen JH, Smith BR, Keijer WJ, van Gijn R, Ven Bokkel Huinink WW, Vlasveld LT, et al.
High performance liquid chromatographic analysis of the new anti-tumor drug SK&F 104864-A (NSC 609699) in plasma.
J. Pharm. Biomed. Anal. 1990;8:789–794. MEDLINE |
CrossRef
[27].
[27]
Vey N, Kantarjian H, Beran M, O’Brien S, Cortes J, Giles FJ, et al.
Combination of topotecan with cytarabine or etoposide in patients with refractory or relapsed acute myeloid leukemia: results of a randomized phase I/II study.
Blood. 1998;92:235a;
. [28].
[28]
Rowe JM, Andersen J, Mazza JJ, Paietta E, Bennett JM, Hayes A, et al.
Phase III randomized placebo-controlled study on granulocyte-macrophase colony stimulating factor (GM-CSF) in adult patients (55–70 years) with acute myelogenous leukemia. (AML): a study of the Eastern Co-operative Oncology Group (ECOG).
Blood. 1995;86:4570462. [29].
[29]
Ohno R, Tonoga M, Kobayashi T, Kanamaru A, Shirakaw S, Masaoka T, et al.
Effect of granulocyte colony stimulating factor after intensive induction therapy in relapsed or refractory acute leukemia.
New Engl. J. Med. 1990;323:871. MEDLINE [30].
[30]
Wall JG, Burris HA, Von Hoff DD, Rodriguez G, Kneuper-Hall R, Shaffer D, et al.
A phase I clinical and pharmacokinetic study of the topoisomerase inhibitor topotecan (SK&F 104864) given as an intravenous bolus every 21 days.
Anticancer Drugs. 1992;4:337–345. [31].
[31]
O’Dwyer PJ, LaCreta FP, Haas NB, Halbherr T, Frucht H, Goosenberg E, Yao KS. Clinical, pharmacokinetic and biological studies of topotecan. Cancer Chemother Pharmacol 1994;34:S46–52. [32].
[32]
Haas NB, LaCreta FP, Walczak J, Hudes GR, Brennan JM, Ozols RF, et al.
Phase I/pharmacokinetic study of topotecan by 24-hour continuous infusion weekly.
Cancer Res. 1994;54:1220–1226. MEDLINE [33].
[33]
Montazeri A, Boucard M, Lokiec F, Pinguet F, Culine S, Deport-Fety R, et al.
Population pharmacokinetics of topotecan: intra-individual variability in total drug.
Cancer Chemother. Pharmacol. 2000;46:375–381. MEDLINE |
CrossRef
[34].
[34]
Van Warmerdam LJC, Creemers G-J, Rodenhuis S, Roding H, deBoer-Dennert M, Schellens JHM, et al.
Pharmacokinetics and pharmacodynamics of topotecan given on a daily times five schedule I phase II clinical trials using a limited sampling procedure.
Cancer Chemother. Pharmacol. 1996;38:254–260. MEDLINE |
CrossRef
[35].
[35]
Furman WL, Baker SD, Pratt CB, Rivera GK, Evans WE, Stewart CF.
Escalating systemic exposure of continuous infusion topotecan in children with recurrent acute leukemia.
J. Clin. Oncol. 1996;14:1504–1511. [36].
[36]
Gallo JM, Laub PB, Rowinsky EK, Grochow LB, Baker SD.
Population pharmacokinetic model for topotecan derived from phase I clinical trials.
J. Clin. Oncol. 2000;18:2459–2467. [37].
[37]
Perego P, Capranico G, Supino R, et al.
Topoisomerase I gene expression and cell sensitivity to camptothecin in human cell lines of different tumor types.
Anticancer Drugs. 1994;5:645–649. MEDLINE |
CrossRef
[38].
[38]
Dubrez L, Goldwasser F, Genne O, Pommier SE.
The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors.
Leukemia. 1995;9:1013–1024. MEDLINE [39].
[39]
Slichenmyer WJ, Rowinsky EK, Donehower RC, Kaufman SH.
The current status of camptothecin analogues as antitumor agents.
J. Natl. Cancer Inst. 1993;85:271–291. MEDLINE [40].
[40]
Potmesil M.
Camptothecins: from bench research to hospital wards.
Cancer Res. 1994;54:1431. MEDLINE [41].
[41]
Pommier Y, Leteurtre F, Fesen MR, et al.
Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors.
Cancer Invest. 1994;12:530. MEDLINE |
CrossRef
[42].
[42]
Kapoor R, Slade DL, Fujimori A, et al.
Altered topoisomerase I expression in two subclones of human CEM leukemia selected for resistance to camptothecin.
Oncol. Res. 1995;7:83–95. MEDLINE [43].
[43]
Fujimori A, Harker WF, Kohlhagen G, et al.
Mutation at the catalytic site of topoisomerase I in CEM/C2 a human leukemia cell line resistant to camptothecin.
Cancer Res. 1995;55:1339–1346. MEDLINE [44].
[44]
Kaufman SH, Svingen PA, Gore SD, et al.
Altered formation of topotecan stabilized topoisomerase I- DNA adducts in human leukemia cells.
Blood. 1997;89:2098–2104. MEDLINE [45].
[45]
Wang LF, Ting CY, Lo CK, et al.
Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance.
Cancer Res. 1977;57:1516–1522. MEDLINE [46].
[46]
Beadler DR, Chang JY, Zhou BS, et al.
Camptothecin resistance involving steps subsequent to the formation of protein linked DNA breaks in human camptothecin-resistant KB cell lines.
Cancer Res. 1996;56:345–353. MEDLINE [47].
[47]
Saleem A, Ibrahim N, Patel M, Li XG, Gupta E, Mendoza J, et al.
Mechanisms of resistance in a human cell line exposed to sequential topoisomerase poisoning.
Cancer Res. 1997;57:5100–5106. MEDLINE [48].
[48]
Grabowski D, Ganapathi R.
Cytotoxic efficacy with combinations of topoisomerase I and topoisomerase II inhibitors in sensitive and multidrug-resistant L1210 mouse leukemia cells.
Ann. NY Acad. Sci. 1996;803:306–307. MEDLINE |
CrossRef
[49].
[49]
Nair JS, Kancherla R, Seiter K, Traganos F, Tse-Dinh YC.
Action of topoisomerase targeting drugs on non-Hodgkin’s lymphoma and leukemia: correlation of clinical and cell culture studies.
Ann. NY Acad. Sci. 2000;922:326–329. MEDLINE [50].
[50]
Whitacre CM, Belfi CA, Berger SJ, Gosky DM, Garcia J, Bao C, Sramkoki M, Berger NA. Cell cycle dependent modulation of topo IIá levels by topotecan in vitro: potential implications on combination therapy with topo I and II inhibitors. Proc. AACR, 1999;40:109 [abstract 723]. [51].
[51]
Estey E, Alakha RC, Hittelman WN, et al.
Cell cycle stage dependent variations in drug induced topoisomerase II mediated DNA cleavage and cytotoxicity.
Biochemistry. 1987;26:4338–4344. [52].
[52]
Del Bino G, Skierski JS, Darzynkiewicz Z.
Diverse effects of camptothecin an inhibitor of topoisomerase I on the cell cycle of lymphocytic (L1210, MOLT-4) and myelogenous (HL-60, KG1) leukemic cells.
Cancer Res. 1990;50:5746–5750. MEDLINE [53].
[53]
Stahl M, Kasinir-Bauer S, Harstrick A.
Down-regulation of topoisomerase II by camptothecin does not prevent additive activity of the topoisomerase II inhibitor etoposide in vitro.
Anticancer Drugs. 1997;8:671–676. MEDLINE |
CrossRef
[54].
[54]
Kaufman SH.
Antagonism between camptothecin and topoisomerase II directed chemotherapeutic agents in a human leukemia cell line.
Cancer Res. 1991;51:1129–1136. MEDLINE [55].
[55]
Vey N, Keating MJ, Rios MB, Estery E.
Effect of CR on survival in patients with AML receiving first salvage therapy.
Blood. 1998;92:235a. [56].
[56]
Gore SD, Rowinsky EK, Miller CB, Griffin C, Chen TL, Borowitz M, et al.
A phase II window study of topotecan in untreated patients with high risk adult acute lymphoblastic leukemia.
Clin. Cancer Res. 1998;4:2677–2689. MEDLINE Departments of Medicine, Biostatistics and Epidemiology, Pharmacology, and Pathology of University Hospitals of Cleveland, Ireland Cancer Center, Case Western Reserve University, Louis Stokes Veterans Affairs Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106, USA  Corresponding author. Tel.: +1-216-844-3213; fax: +1-216-844-5449.
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