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Activity and Pharmacodynamics of 21-Day Topotecan Infusion in Patients With Ovarian Cancer Previously Treated With Platinum-Based Chemotherapy

From the Kaplan Cancer Center and New York University Medical Center, New York, and Albert Einstein College of Medicine, Bronx, NY.

Address reprint requests to Howard Hochster, MD, 160 E 32nd St, New York, NY 10016; email howard.hochster{at}med.nyu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Twenty-one–day topotecan infusion was administered as second-line therapy in patients with previously treated ovarian cancer (based on our prior favorable phase I experience) to determine its activity, time to progression, and pharmacodynamics.

PATIENTS AND METHODS: Ovarian cancer patients with measurable lesions and one prior platinum-containing regimen were eligible. Topotecan 0.4 mg/m²/d 21-day continuous ambulatory intravenous infusion, with appropriate dose modifications for toxicity, was administered every 28 days. Weekly blood levels of topotecan and topoisomerase-1 (topo-1) levels in peripheral-blood mononuclear cells (PBMCs) were determined for pharmacodynamic correlation.

RESULTS: Twenty-four patients were entered onto the study (six cisplatin-refractory, five relapsing within < 6 months and 13 relapsing > 6 months after platinum-based therapy). A total of 128 cycles of topotecan (median, four cycles per patient; range, one to 12 cycles) were administered. The major toxicity was neutropenia (29% grade 3 in all cycles and 4% grade 4). One episode of grade 4 thrombocytopenia (4%) occurred. Fifty-two percent of the patients had anemia that required transfusions. Eight of 23 patients with measurable disease (35%; 95% confidence interval [CI], 15% to 54%) had partial responses (PRs) lasting longer than 1 month. Two of these patients had minor residual computed tomographic changes but had clinical complete remissions that lasted up to 53 weeks while they were not undergoing further therapy. One patient with nonmeasurable disease had a PR (by CA-125 criteria) that lasted 6 months, for an overall response rate of 38% in nine of 24 patients (95% CI, 18% to 57%). The median time to progression was 26 weeks. Pharmacodynamic analysis demonstrated a statistically significant decrease in free PBMC topo-1 level at weeks 2 and 3 of drug administration. There was a strong statistical correlation between the decrease in free topo-1 levels and increasing area under the curve (AUC) for topotecan. This was confirmed in a pharmacodynamic model.

CONCLUSION: Twenty-one–day infusion is a well-tolerated method of administering topotecan. Pharmacodynamic studies demonstrate correlations between (1) the week of infusion and the PBMC topo-1 level, (2) the AUC of topotecan and the decrease in topo-1 levels, and (3) the change in topo-1 level and the neutrophil nadir. The objective response rate of 35% to 38% (95% CI, 15% to 57%) in this small multicenter study is at the upper level for topotecan therapy in previously treated ovarian cancer. Prolonged topotecan administration therefore warrants further investigation in larger, randomized studies comparing this 21-day schedule with the once-daily-for-5-days schedule.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
OVARIAN CANCER CONTINUES to be a major cause of morbidity and mortality. It is estimated that 26,700 new cases of ovarian cancer are diagnosed in the United States yearly and that nearly 15,000 women will die, making this tumor the leading cause of gynecologic cancer death.¹ The majority of patients present with stage III and IV disease and require chemotherapy for residual postoperative disease and potential cure. Alkylating agents were initially used as single agents in this setting, with objective response rates of 33% to 65% and median survival of 10 to 14 months.² In prospective randomized studies, combination chemotherapy, which included a platinum compound combined with other agents, was found to be superior to both single agents and to nonplatinum-containing multidrug regimens.³ With modern combination chemotherapy regimens, complete response rates in first-line therapy are reported to be 25% to 35%, with median survivals of up to 38 months in patients with suboptimally debulked tumors and up to 53 months for optimal patients.⁴ Most recently, paclitaxel became a first-line therapy method for this disease, on the basis of the randomized studies of cisplatin plus paclitaxel, compared with cisplatin plus cyclophosphamide,^4,5 and is frequently used with either cisplatin or carboplatin as initial therapy. Despite these advances, the majority of patients with ovarian cancer presenting with stage III and IV disease will relapse and require additional therapy. The use of newer agents in the development of potentially more active treatment regimens is extremely important.

Topoisomerase-1 (topo-1) inhibitors are newer agents that show promising activity in the treatment of ovarian cancer. Topo-1, the cellular target for these agents, is a 100-kd intranuclear enzyme that is intimately involved with cell replication and transcription, specifically through DNA-unwinding mechanisms.^6-10 The natural plant product camptothecin and its semisynthetic derivatives, including topotecan, have been shown to bind to topo-1, leading to single-stranded, protein-associated DNA breakage and cellular cytotoxicity. Topotecan, a semisynthetic analog of camptothecin, incorporates a 10-hydroxyl substitution on the A-ring and a tertiary amine at the 9 position. These chemical modifications achieve the dual goals of increasing the water solubility of topotecan, compared with that of the parent compound, and stabilizing the lactone form of the E-ring that is necessary for biologic activity. Prior animal studies using a less water soluble camptothecin analog (9-amino-camptothecin) suggested that continuous depot injection yielded a curative level of activity against colonic xenografts.¹¹ Preclinical in vivo studies of topotecan also demonstrate schedule dependency.

We conducted a phase I study of topotecan, sponsored by The National Cancer Institute's Cancer Therapy Evaluation Program, to test the tolerability of continuous ambulatory infusion beginning at a dose of 0.2 mg/m²/d for 7 days and eventually escalating to 21 days if no toxicity was evident. The maximum-tolerated dose for this regimen was determined to be 0.53 mg/m²/d for 21 days.¹² Furthermore, in this phase I study, three of six patients with ovarian cancer who had been heavily pretreated with chemotherapy including paclitaxel demonstrated partial responses when treated with prolonged topotecan infusion. A phase II study was therefore initiated by the New York Gynecologic Oncology Group (NYGOG) to more fully investigate the activity of prolonged topotecan infusion in ovarian cancer.

Additional studies with topotecan infusions suggested that peripheral-blood mononuclear cell (PBMC) topo-1 levels correlated with pharmacokinetic parameters and toxicity.^13,14 In these studies, we developed the Western blot method as a sensitive method of measuring changes in topo-1 levels during exposure to topotecan (or potentially to any topo-1 inhibitor compound). We were able to successfully determine serial topo-1 values for noncomplexed ("free") topo-1, which diminishes with topotecan treatment, but could not reproducibly measure complexed topo-1 because of the smaller amounts present and the increased degradation during processing by activated proteases. In these preliminary studies, we showed that PBMC topo-1 levels decreased during infusion, but we could not correlate these changes with toxicity because of the phase I design and the variation in extent of prior treatment.

We had a secondary objective of assessing the pharmacodynamics of prolonged topotecan infusion in this more defined group of patients who were treated uniformly, using PBMCs as a readily accessible and easily obtainable source of normal tissue for repeated sampling in the course of prolonged administration of a topo-1 inhibitor.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
Eligibility requirements for participation in this study included histologic documentation of metastatic epithelial ovarian cancer in women who had previously been treated with one, and only one, platinum-containing chemotherapy regimen. Patients were grouped by prior response to platinum: (1) platinum-refractory patients, whose disease progressed or who remained stable on initial platinum-based chemotherapy; (2) platinum-resistant patients, who responded but subsequently relapsed within 6 months of their discontinuing initial platinum-based chemotherapy; or (3) platinum-sensitive patients, who responded but subsequently relapsed more than 6 months after discontinuing initial platinum therapy. At least 4 weeks had to have elapsed since the date of prior surgery or last chemotherapy treatment. A semipermanent venous access device was required. All patients were required to have bidimensionally measurable disease on physical examination, abdominal-pelvic computed tomography (CT) scan, magnetic resonance imaging, ultrasound, or chest x-ray including at least one tumor with a diameter 2 cm. Eligible patients were required to have the following laboratory values: hemoglobin 9.0 mg/dL; WBC count, 3.5 x 10³/µL; granulocyte count 1,500/µL; platelet count 100 x 10³/µL; and creatinine 1.5 mg/dL or creatinine clearance rate 60 mL/min. The serum bilirubin level was required to be 2.0 mg/dL and transaminase level less than five times the upper limit of normal. An Eastern Cooperative Oncology Group (ECOG) performance status less than or equal to 2, with life expectancy of at least 3 months, was required. All patients gave written informed consent to participate in this protocol, as approved by the institutional review boards at each participating institution.

Treatment Details
Patients were initially treated with a topotecan dose of 0.4 mg/m²/d for 21 days. The drug was administered by ambulatory infusion pump via venous access device at an infusion rate of 7 mL/d (49 mL/wk), allowing for infusion pump cassette changes once each week. Treatment cycles were repeated every 28 days as long as the neutrophil count was 1,500/µL and the platelet count was 100,000/µL on day 28. If not, the course was delayed 1 week and the topotecan dose in the next cycle was reduced by 0.1 mg/m²/d. Infusions were discontinued if the platelet count was less than 50,000/µL, the WBC count was less than 1,000/µL, or the absolute neutrophil count (ANC) was less than 500/µL at any time during the 21-day infusion. The topotecan dose in the next cycle was also reduced by a level of 0.1 mg/m²/d for patients who had an ANC nadir of less than 500/µL, a platelet nadir of less than 50,000/µL, or nonhematologic grade 3 or 4 toxicity. In patients having no greater than grade 2 hematologic or nonhematologic toxicity in any cycle, the topotecan dose could be escalated by 0.1 mg/m²/d in subsequent cycles. No granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor treatment was allowed during the topotecan infusion. Patients were assessed with weekly complete blood cell counts, monthly CA-125 tests, and abdominal-pelvic CT scans every two cycles during therapy. Complete response was defined as complete resolution of all evidence of disease on CT scan and physical examination, with normalization of CA-125 that lasted at least 1 month. Partial response was defined as a greater than 50% decrease in the sum of the products of the greatest length and perpendicular measurements of all measurable lesions for at least 1 month, without the appearance of new lesions.

Mononuclear Cell Preparation
Mononuclear cells were prepared from two heparinized tubes, each containing 20 mL of blood, using standard methods¹⁵ with the following modifications to allow for topotecan measurement and the stabilization of the mononuclear preparation for Western blot analysis. The tubes were centrifuged at 1,000 x g for 10 minutes to separate the plasma from the buffy coat. The separated plasma was stored frozen at -20°C. The remaining buffy coat from each of the heparinized tubes was diluted with 5 mL of RPMI 1640 medium and then processed through a standard Ficoll-Hypaque gradient centrifugation step.^15,16 The mononuclear cell layer was removed, washed twice with phosphate-buffered saline, and centrifuged at 1000 x g. The cells were resuspended in a volume of 0.3 mL, and 0.05 mL was removed and frozen for the DNA analysis. We then added 0.25 mL of 2x sodium dodecyl sulfate sample buffer (20% glycerol; 10% beta-mercaptoethanol; 6% sodium dodecyl sulfate; 125 mM Tris, pH 6.8; bromphenol blue 0.2%) to the remaining cell suspension and mixed the sample. This portion of the sample was then boiled for 5 minutes at 100°C and stored at -70°C until it was processed for Western blot analysis.

Pharmacokinetics
Plasma specimens were processed according to our modifications of a previously described method using methanol dilution.¹⁷ To allow for better sensitivity, given the low doses administered, we used a solid-phase extraction (SPE) technique that concentrated, rather than diluted, the specimens in the protein-removal step. Specimens were processed using C-18 SPE columns as previously described, with acidification of the extracted plasma to allow for conversion of all of the topotecan to the lactone form, followed by high-performance liquid chromatography reverse-phase chromatographic analysis as detailed below.

Three to 5 mL of plasma from clinical samples were centrifuged at 1000 x g for 10 minutes; buffers and an internal standard (SKF 105107; SmithKline Beecham, King of Prussia, PA) were then added. The reference internal standard was camptothecin, kindly provided by Dr. Monroe Wall (Research Triangle Park, NC). The camptothecin was dissolved in 0.1 M NaH₂PO₄, pH 3.5 (concentration, 1 mg/mL), which was added in sufficient quantity to yield a final concentration of 10 ng/mL of undiluted plasma before the application of the plasma to the SPE extraction columns (Bond-elute; Varian, Torrance, CA). Topotecan and the internal standard were then eluted from the column within 5 minutes. A standard set of plasma containing topotecan in concentrations of 2.5 to 100 ng/mL was processed and analyzed with each clinical specimen to provide a standard curve. Typically, for the analysis of the total topotecan content, 0.2 mL of the extract was mixed with 0.1 mL of 0.05M H₃PO₄, allowed to sit at room temperature for 1 hour, and then analyzed for total drug content with the injection of 50 to 100 µL of the sample into the high-performance liquid chromatography apparatus. The high-performance liquid chromatography system used a 15 cm x 4.6 mm 3-µm C-18 column (Phenomenex, Torrance, CA) conditioned with 67% methanol containing 0.01M dioctyl sodium succinate, 1 mM NH₂PO₄ (pH 6.0), and 0.3% triethylamine at a flow rate of 1 mL/min. Fluorescence detection was performed using an Applied Biosystems 970 fluorometer (Perkin-Elmer, Norwalk, CT) set at 380-nm excitation with a 470-nm cutoff filter at 0.01 µA full scale. The chromatographic system consisted of a Knauer model 42 pump (Sonntek, Inc., Upper Saddle River, NJ) and a Waters model 712 WISP autosampler (Waters Associates, Milford, MA) controlled by an Axxiom model 747 PC controller/data system (Axxiom Chromatography Inc, Moorpark, CA). Each sample was run in duplicate, and the results were expressed as an average of the values. Samples reflecting the attainment of steady-state levels (day 7 and day 15) were averaged together for use in the area under the curve (AUC) correlations. Blood levels were modeled using WinNonlin Version 4.5 (Scientific Consulting Inc, Apex, NC) with a steady-state infusion model.

Topo-1 Determination by Western Blot
Protein mixtures obtained from the cell lysates were separated by 6% polyacrylamide gel electrophoresis, with 50 µL of each sample loaded per well. Duplicate analyses were run for each sample, and an adequate sample amount was left over for reanalysis at a later date if necessary. The Western blot methodology was essentially performed as was detailed recently.¹⁴

DNA Analysis
PBMC aliquots from the same samples as those used in the Western blot assay were analyzed for DNA content, using a modification of the diphenylamine colorimetric determination¹⁸ for use in a microtiter plate reader. Three hundred–microliter aliquots were used in each well, with the absorbance determined at 570 nm.

Antibody Procurement
Serum samples for SCL-70 antibody screening were obtained from patients with scleroderma.¹⁹ To obtain a supply of sera rich in the SCL-70 antibody, a panel of the patients' serum specimens were analyzed by Western blot with varying concentrations of HeLa cell extracts, which are known to have a high level of topo-1 expression.²⁰ Comparisons made between various samples yielded a patient with a high titer of highly specific SCL-70 antibody. Aliquots of 80 µL of this serum were stored at -80°C. This reagent was used for all studies in which we report the topo-1 protein copy numbers.

Statistical Methods
This study was initially conceived as a multicenter phase II study sponsored by SmithKline Beecham to be conducted in the NYGOG and several international sites. The study design mandated a total accrual of 36 patients (18 from NYGOG and 18 internationally). The study was stopped after additional accrual at NYGOG sites and limited accrual internationally. We report our results on the basis of the analysis of the 24 patients accrued within the NYGOG sites.

Paired analyses were performed using the Wilcoxon rank sum test or signed rank test procedures. Weekly cycles were compared with respect to topo-1 baseline values and to topo-1 values at weeks 1, 2, 3, and 4. Within each set, appropriate adjustments of the P value for significance were made using the Bonferroni multiple comparison procedure. Correlation descriptions and hypothesis testing were performed using the Spearman procedure. Comparison by individuals between cycles were performed using paired procedures (Wilcoxon rank sum or signed rank tests). Testing between groups used the log-rank procedure (JMP; SAS, Research Triangle Park, NC). Time to progression was estimated by the method of Kaplan and Meier,²¹ using Statview software (Research Triangle Park, NC). Pharmacodynamic modeling was carried out using prepared data sets of PBMC topo-1 levels, AUCs for the plasma topotecan concentrations, and complete blood cell counts obtained at baseline and at the time of determination of the WBC, ANC, and platelet nadir values. These data sets were analyzed using a maximal effect (E_max)–inhibitory effect model available in WinNonlin Version 4.5 (Scientific Consulting Inc).

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Twenty-four patients were entered onto the study and were assessable for response and toxicity. Patient demographics are listed in Table 1. The median age of all 24 patients was 56 years (range, 34 to 83 years). The majority of patients had papillary serous adenocarcinoma (19 of 24). Thirteen patients had an ECOG performance status of 0, and 10 patients had an ECOG performance status of 1. Six patients were refractory to initial therapy, five had an early relapse (within 6 months of completing initial chemotherapy), and 13 had a late relapse (more than 6 months). Prior therapy for these 24 patients included paclitaxel plus carboplatin (ten patients), cyclophosphamide plus carboplatin (ten patients), paclitaxel plus cisplatin (three patients), and cyclophosphamide plus cisplatin (one patient). Twenty-three of the 24 patients had measurable disease, on the basis of a physical examination or a CT scan showing at least one lesion greater than 2.0 cm in diameter. One patient, who had an elevated CA-125 level at baseline and assessable disease on laparoscopy, was entered onto the study at her physician's request (despite her not meeting the standard eligibility criteria), with the agreement that she would be reassessed with follow-up laparoscopy.

Toxicity
A total of 128 cycles of topotecan infusion were administered, and the number of cycles administered per patient ranged from one to 12 (Table 2). Eleven of the 24 patients received more than six cycles of topotecan infusion. No patient developed grade 4 hematologic toxicity in the first cycle. Six patients received dose escalations to 0.5 mg/m²/d, on the basis of their having had grade 2 toxicity or less in the first cycle. Fifteen cycles were administered at this dose level. Fourteen patients required a dose reduction at some point in their therapy because of their failure to achieve hematologic recovery within 28 days (12 patients) or the interruption of therapy because of low blood cell counts before day 21 (two patients). Forty-four cycles were administered at reduced doses, including 33 cycles at 0.3 mg/m²/d.

Seven patients (28%) developed grade 3 neutropenia as a maximum hematologic toxicity and one patient (4%) developed grade 4 neutropenia, which corresponded to four episodes of grade 3 leukopenia (16%) without grade 4 leukopenia (Table 3). One patient developed grade 4 thrombocytopenia. Thirteen patients (52%) required RBC transfusions during the course of therapy, and five patients received erythropoietin. Use of colony-stimulating factors was not needed.

Nonhematologic toxicity was generally mild and not dose-limiting. Approximately one third of patients reported a maximum of grade 1 alopecia or fatigue. Nausea and vomiting of grade 1 severity was reported in nine patients and was of grade 2 severity in three patients.

Response
Eight of the 23 patients (35%; 95% confidence interval [CI], 15% to 54%) with measurable disease had objective partial responses, which included greater than 50% tumor shrinkage that lasted more than 1 month (Table 4). By CT scan criteria, seven patients responded. All responses were reviewed by an independent radiologist and confirmed as partial responses (Scott Fields, MD, personal communication, 1998). One patient with a 6-cm cutaneous plaque responded with a more than 50% tumor shrinkage on physical examination in the first cycle, with the response lasting for three cycles. One additional patient had a normal CT scan with peritoneal nodules approximately 0.5 cm in size observable on laparoscopy. Her CA-125 level was 194 at baseline and returned to normal in the first cycle. The drop persisted until the sixth cycle, when she progressed with new disease on CT scan (as well as an increasing CA-125 level). By CA-125 criteria, she is considered a partial responder. The responders included two of six patients with platinum-refractory disease, two of five with early relapse, and five of 13 with late relapse (Table 5). Additionally, the cases of two patients with near complete responses and normalization of CA-125 but minimal, persistent CT scan abnormalities were classified as partial responses. Clinically, however, they were considered by their treating physicians to have had complete responses at the time of therapy and had not undergone any further therapy for 6 and 8 months before relapsing. Median time to progression (calculated by the method of Kaplan and Meier²¹) for all patients entered onto the study was 26 weeks, with a range of 8 to 60 weeks (Fig 1).

View larger version (10K): [in this window] [in a new window]   Fig 1. Probability of freedom from progression (weeks), by the method of Kaplan and Meier.21 (——), Cumulative survival; (), event times; (•), censor times.

Pharmacodynamic Analyses
Fifty-eight samples for the determination of steady-state levels of plasma topotecan concentration and PBMCs for topo-1 levels were obtained from 21 patients. Distributional characteristics were evaluated for all pharmacodynamic results and were uniformly found to be nonnormally distributed. Consequently, all descriptions and hypothesis testing will be presented using ranks and nonparametric statistics. Figure 2 presents the weekly values of free topo-1 in PBMCs during cycles 1 and 2. This figure demonstrates that within each cycle, there is a decrease in the topo-1 level during topotecan administration. For cycles 1 and 2, there is a decrease in the PBMC topo-1 level in apparent response to the duration of treatment. In these two cycles, the results for week 3 of each cycle seem to plateau, compared with the results for week 2 (comparative values not significantly different), although the topo-1 levels return to the baseline values once the infusion is stopped. For example, in the first cycle, the topo-1 level starts with a median baseline value of 8.0 x 10⁵ copies/cell, decreases to 6.2 (week 1) and then 3.8 (week 2), increases to 5.0 at week 3, and returns to baseline (or slightly higher) at 10.8 x 10⁵ copies/cell by week 4, 1 week after drug treatment has stopped. The values for these weekly levels of PBMC topo-1 showed statistically significant decreases for weeks 1, 2, and 3, compared with the baseline values (for cycle 1: baseline v week 1, P < .001; baseline v week 2, P < .001; baseline v week 3, P < .001; signed rank test with Bonferroni criterion for multiple comparisons requires P < .025 for significance). In cycle 2, the only difference which approached significance was that between baseline for the second cycle (week 4) and week 5 (P < .04). The values for weeks 6 and 7 were not significantly different from those for week 4 (P = .06 and .46, respectively). Within the weeks of topotecan infusion (weeks 1, 2, 3, and 4, 5, 6), the only difference in the values of topo-1 which approached statistical significance was that between week 1 and week 2 (P = .034), although it does not meet the Bonferroni criterion.

View larger version (8K): [in this window] [in a new window]   Fig 2. Weekly PBMC topo-1 levels during the first two cycles of therapy (weeks 0 through 7) (mean [x105 copies/cell) ± SEM and individual values]. Week 1, 2, and 3 levels had decreased from baseline (all P < .001, signed rank test, Bonferroni correction for multiple comparisons requiring P < .025).

The relationship between PBMC topo-1 level and topotecan administration also fits a pharmacodynamic model based on enzyme inhibition kinetics (the E_max inhibitory effect model). This is demonstrated in Fig 3, which shows topo-1 level as a function of total topotecan AUC and includes the curve of the model, together with the two 90% CI curves. The AUC was calculated from day 1 of the cycle to the time point sampled or the end of topotecan infusion and was modeled on the basis of the measured steady-state concentrations. The points (n = 58) represented in the figure show all of the weekly data for 21 patients sampled during the first cycle of therapy. The E_max model yields a regression equation with an average coefficient of variation of 15% for the parameter estimates. This model is consistent with the current knowledge of the mechanism of topo-1 inhibition by topotecan and the changes in PBMC topo-1 levels described above.

View larger version (13K): [in this window] [in a new window]   Fig 3. Pharmacodynamics of PBMC topo-1 level versus total topotecan AUC (ng-h/mL) (58 weekly blood values in 21 patients; first cycle of therapy only). The Emax inhibitory effect model with the 90th percentile CI curves is shown (average coefficient of variation = 15% for Emax and median effective concentration (EC50) parameters). (), Topotecan level; (——), predicted topotecan level; (), universal Emax, upper; (), universal Emax, lower; (x), EC50, lower; (x), EC50, upper.

Finally, we investigated the relationship of topo-1 level in PBMCs with hematologic toxicity. For each cycle, the difference between the baseline topo-1 level and the nadir of the topo-1 level (delta-topo) for that cycle was examined. We also examined the changes from baseline to nadir values for ANC, WBC and platelets in each cycle. We also investigated the relationship between the AUC of the topotecan drug level with hematologic parameters and the AUC of the topo-1 determinations with hematologic nadirs and the AUC for weekly blood cell counts. The best results were obtained using the differences between baseline and nadir values.

A Spearman correlation was performed to evaluate the magnitude and direction of the association between delta-topo and delta-ANC, delta-WBC, and delta-PLT for each cycle. An association (Spearman rho = 0.657; P < .02) was found for the relationship between change in ANC and change in topo-1 level in cycle 2. Because a Bonferroni procedure was used, necessitating a value of P = .025 for significance, this finding showed statistical significance. In our data set, there were two extreme values of the initial ANC that were likely to influence the Spearman statistic for correlation with delta-ANC in the first cycle, and, therefore, the relationship could not be adequately defined for cycle 1. No effects were seen for the correlation of delta-WBC with delta-topo. Because of the low incidence of thrombocytopenia, we could not adequately examine the relationship between change in topo-1 level and platelet nadir.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Prior experimental evidence has suggested that the observed antitumor activity of topo-1 inhibitors is schedule-dependent. Multiple-day administration schedules have shown greater activity than single-dose schedules for topotecan (Randall Johnson, PhD, data on file, SmithKline Beecham) and other camptothecin analogs.⁹ Because of these considerations, we previously performed a phase I study that escalated the duration of topotecan infusion from 7 to 21 days every 28 days. We saw promising activity with prolonged infusion of topotecan in women with ovarian cancer who had previously been treated with multiple active agents, including paclitaxel.

We therefore began this multiinstitutional study in the NYGOG to investigate the activity of topotecan given by the prolonged infusion schedule. The eligibility criteria for this study were identical to those used in the multicenter international studies of topotecan given by 5-day bolus schedule. The response rate for topotecan was noted to be 16% for patients treated previously with a platinum-containing regimen in the multicenter phase II study²² and 20.5% in the randomized study.²³ Of additional interest is the level of toxicity observed using topotecan at a dose of 1.5 mg/m²/d in the 5-day bolus schedule. In the randomized study, 80% of patients experienced grade 4 neutropenia; an additional 15%, grade 3 neutropenia; and 25%, grade 4 thrombocytopenia. Also, the high incidence of toxicity with the once-daily-for-5-days regimen (1.5 mg/m²/d) resulted in early termination of the phase II ECOG endometrial cancer study.

In this study, we observed a high response rate of 35% (eight of 23; 95% CI, 15% to 54%) in those patients with measurable disease or 38% (nine of 24), including the one patient with laparoscopic disease and CA-125 evidence of response. All seven CT responses were reviewed and confirmed by independent radiologists in a fashion similar to that in the larger, daily-for-5-days study. Responding patients included those with platinum-refractory disease (two of six), early relapsers (two of five), and late relapsers (five of 13 patients). A similar study was carried out by British investigators, using the 21-day infusion schedule, who preliminarily reported a lower response rate of 11%, with an additional 17% of patients achieving disease stabilization.²⁴ The difference in results may be due to selection factors and a greater number of platinum-resistant patients in the European study or the early declaration of disease progression (Robert Beckman, MD, personal communication, 1998). Whether the higher response rate observed in this study, compared with that of the bolus schedule, is due to greater activity of the prolonged administration schedule of topotecan or to other factors (including selection factors or beta-error) cannot be determined without direct, randomized, comparative studies. The median time to progression of 26 weeks in this study compares favorably with the 23-week median time to progression for topotecan in the randomized study and 14 weeks for paclitaxel in the same study.²³

The pharmacodynamic studies that we have performed support the hypothesis that prolonged administration of topotecan has a mechanistic basis which could result in greater antitumor activity. We have demonstrated a direct relationship between the AUC for topotecan administration and the decrease of free topo-1 in PBMCs sampled weekly during treatment. This relationship fits an accepted model of pharmacodynamic interaction. The time course of these studies suggests inhibition of topo-1 for the duration of drug infusion, with recovery when the drug treatment is stopped. We have also demonstrated a relationship between increasing hematologic toxicity (neutropenia) and topo-1 depletion. We are continuing these studies and believe that using newer methods to preserve PBMC viability (using Vacutainer CPT tubes [Becton Dickinson and Company, Franklin Lakes, NJ]) may reduce some of the systematic variability in the topo-1 level measurement.

On the basis of the promising activity for prolonged topotecan administration observed in this trial, the long survival time, the more favorable toxicity profile for this schedule, and the supporting pharmacodynamic studies of topo-1, this schedule of administration deserves further investigation in the treatment of ovarian cancer. Its more favorable toxicity profile, compared with that of the 5-day bolus schedule, suggests that it may be more easily combined with other agents. We are currently extending the observations reported here in a study of prolonged topotecan infusion with cisplatin as first-line therapy for women with ovarian cancer. In addition, with the availability of oral topotecan, the prolonged administration of this topo-1 inhibitor will be more feasible and is currently under investigation in combination regimens.

	ACKNOWLEDGMENTS

 
Supported in part by National Institutes of Health grants no. CA 16087, RO-1 CA 56129, and UO-1 CA 63422, and by a grant from SmithKline Beecham Pharmaceuticals

We thank Tony Kim and David Fry for their dedicated laboratory efforts; Anne Jacquotte, MD, PhD; and our hard-working nurses Lorraine Centrilla, RN, Rosalie Odchimar-Reissig, RN, and Hilda Haynes, RN.

We dedicate this manuscript to our friend and colleague, Julia Zelmanovich, MD, most recently a resident in the Department of Obstetrics and Gynecology at the Albert Einstein College of Medicine, who had contributed heavily to the pharmacodynamic analysis presented here and recently lost her life in a tragic motor vehicle accident.

	NOTES

 
Presented in part at the American Society of Clinical Oncology Meeting, Philadelphia, PA, May 18-21, 1996, and ECCO-8, September 1996.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
1. Parker SL, Tong T, Bolden S, Wingo PA: Cancer statistics, 1996. CA Cancer J Clin46:5-27, 1996[Abstract]

2. Young RC, Hubbard SP, De Vita VT: The chemotherapy of ovarian cancer. Cancer Treat Rep1:99-110, 1974

3. Ozols RF, Schwartz PE, Eifel PJ: Ovarian cancer, fallopian tube carcinoma and peritoneal carcinoma, in De Vita VT, Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology (ed 3). Philadelphia, PA, Lippincott-Raven, 1997, pp 1502-1539

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Submitted November 30, 1998; accepted April 6, 1999.

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