1104

Topotecan in Chronic Lymphocytic Leukemia Susan O’Brien, M.D., Hagop Kantarjian, M.D., A m y Ellis, Ph.D., Leonard Zwelling, M.D., Elihu Estey, M.D., and Michael Keating, M.B., Ch.B.

Background. Topotecan is one of a new class of agents that targets topoisomerase I (Topo I) and stabilizes the DNA-topol complex, ultimately resulting in cell death. The rationale for the use of topotecan in chronic lymphocytic leukemia (CLL) is based on the finding that levels of Top0 I are elevated in the lymphocytes of pa- tients with this disease.

Methods. Twelve patients with CLL were treated with topotecan to assess its clinical efficacy; in addition, DNA-protein cross-linking was measured after exposure of the cells to topotecan, in an attempt to correlate poten- tial anti-CLL effects with this parameter. The median age of the patients was 63 years; all had received prior ther- apy with fludarabine and four (33%) were resistant to fludarabine. Four patients (33%) had also received ther- apy with chlorambucil, and three of them were resistant to this agent. Seven patients (58%) were Rai stage 1-11 and five (42%) were Rai stage 111-IV. Topotecan was given as a 30-minute infusion at a daily dose of 2 mg/m2 for 5 days, and courses were repeated monthly. Cells were obtained from the patients before treatment and exposed to 2 pM topotecan or control in vitro. In addition, cells were ob- tained from patients after they received the first dose of topotecan and protein-bound DNA was measured with the same technique.

Results. No patient respoAded to therapy with topo- tecan (95% confidence interval, 0-27%). Nonhematologic toxicity was mild and thrombocytopenia occurred in four of eight patients whose conditions could be evaluated. One patient died of myocardial infarction and another died of fungal pneumonia. DNA-protein cross-linking was detected in all nine patients whose cells were as- sessed in vitro, with levels of cleavable complex ranging from twofold to 7.5-fold that of the control cells. Only two

cross-linking detectable in circulating cells after the $ad rst of eight patients with evaluable conditions had incre

From the Departments of Hematology and Clinical Investiga- tion, The University of Texas M. D. Anderson Cancer Center, Hous- ton, Texas 77030.

Address for reprints: Susan O’Brien, M.D., Department of He- matology, Box 61, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

Received August 8, 1994; revision received November 7, 1994; accepted November 7, 1994.

dose of topotecan, which was consistent with the drug’s lack of effect in vivo.

Conclusions. Although exposure of cells from pa- tients with CLL to topotecan in vitro at 2 pM resulted in detectable protein-DNA cross-linking, this effect was not seen in patients who received a bolus dose of 2 mg/m2, and no remissions were noted in 12 patients. Cancer 1995: 75:1104-8.

Key words: topotecan, chronic lymphocytic leukemia, to- poisomerase I, DNA-protein cross-links.

Introduction

The discovery of drugs that react with the nuclear en- zyme topoisomerase I (Topo I) has led to development of a new class of antineoplastic agents. Although the prototype, camptothecin, had activity in preclinical test- ing, it proved to be overly toxic in Phase I clinical tri- als.’,’ Topotecan is a water-soluble analogue of camp- tothecin that interacts with and stabilizes the Top0 I-DNA complex during the enzyme’s strand passing ac- tion, inhibiting Top0 I function3 This inhibition is a crit- ical step in the tumoricidal action of topotecan.

In preclinical models, topotecan has significant ac- tivity in a broad spectrum of tumors, and Phase I trials have determined schedules that can be given with ac- ceptable t~xici ty .~ The investigation of topotecan in chronic lymphocytic leukemia (CLL) was appealing be- cause CLL lymphocytes have elevated levels of Top0 I,5 which may make them vulnerable to agents that inhibit Top0 I, such as topotecan. In addition, topotecan is not as affected by the multidrug resistant pump, p170, as topoisomerase 11-reactive agents.6 Thus, even in pa- tients who have received extensive prior therapy, this drug, with its novel mechanism of action, may have ac- tivity. The drug-stabilized Top0 I-DNA complex can be quantified, because denaturation of this complex leads to permanent cross-linking between the enzyme and the DNA. In addition to assessing the efficacy of topo- tecan in patients with CLL, we measured the formation

Topotecan in CLL/OBrien et al. 1105

of DNA-protein cross-links both in vitro and in vivo in cells from these patients after exposure to topotecan, in an attempt to correlate possible anti-CLL effects with this parameter.

Materials and Methods

Study Population

Twelve patients with CLL were treated after written in- formed consent was obtained. Criteria for entry into the study required the patient to have a Zubrod perfor- mance status of 2 or lower, and normal hepatic and re- nal function^.^

Therapy

Topotecan was supplied by the National Cancer Insti- tute and given as a 30-minute infusion at a daily dose of 2 mg/m2 for 5 days on an outpatient basis. Courses were repeated at 28-day intervals, and responding pa- tients continued on therapy until the maximal response was achieved. Four patients (33%) received 1 course; 7 patients (58%), 2 courses; and 1 patient (8%), 3 courses.

Response Criteria

Response criteria were those defined by the National Cancer Institute (NCI) Working Group for CLL.8 Com- plete remission required the following: complete reso- lution of palpable adenopathy and hepatosplenomeg- aly; neutrophil count, >15OO/pl; platelet count, ~ 1 O O , O O O / p 1 ; hemoglobin level, 21 1.0 g/dl; lympho- cyte count, <4,OOO/pl; and bone marrow aspirate, 130% lymphocytes. Partial remission required a 250% decrease in all lymph node-bearing areas and organ en- largement and a 250% improvement in the baseline of any abnormal hematologic parameters. Bone marrow assessment was not required for partial remission.

Measurements of Top0 I-DNA Complex Formation in Leukemia Cells

The assay for detection of Top0 I-DNA complexes was based on data showing that protein-bound DNA is re- tained on nitrocellulose filters when applied in a buffer of low ionic strength. The filter-bound DNA can be quantified by hybridization to a radiolabeled Alu DNA probe. Details on the development and the validation of this assay in HL-60 leukemia cells have been pre- sented and Ten milliliters of blood was drawn from patients with circulating leukemic cells be- fore chemotherapy with topotecan. Cells were sepa- rated from plasma by centrifugation 250 g and resus-

pended in phosphate-buff ered saline. Leukemic cells were isolated on Ficoll-Hypaque (5.7% Ficoll, 9% ditro- zoate sodium (Pharmacia, Piscataway, NJ) and resus- pended to 5 X 105/mL in Iscove's modified Dulbecco's medium (JHR Biochemicals, Lenexa, KS) before treat- ment with 2 pmol/L topotecan or vehicle (deionized water) for 1 hour at 37OC, in 5% COz.

After drug treatment, 1 X l o6 cells were centrifuged at 13,000 g for 1 minute (in triplicate) and the medium was aspirated. The cells were immediately lysed with 100 pL 1.25% sodium dodecyl sulfate/5 mmol/L, EGTA, pH 8, at 65OC. Lysates were vigorously vortexed and incubated at 65OC for 10 minutes before DNA was sheared with a 27-gauge needle. One milliliter of 65O protein-binding buffer (0.4 guanidine HCl; 10 mmol/l Tris, pH 8; 10 mmol/l EGTA, pH 8; 0.01% Sarkosyl; 0.3 mol/L NaCl; 10 mmol/l MgCl,) was added to the lysed cells. These lysates were applied to nitrocellulose filters by using a dot blotter (both from Schleicher and Scheull, Keene, NH). Under the buffer conditions, only protein-bound DNA was retained on the filter. DNA was fixed to the nitrocellulose filter by baking at 8OoC for 2 hours in a vacuum oven.

The amount of DNA on a filter was determined by hybridization to Alu DNA (provided by Dr. Grady Saunders, M.D. Anderson Cancer Center, Houston, TX) that had been radiolabeled by random priming with [32P]dCTP (Amersham, Arlington Heights, IL) using the Amersham Multiprime Labeling System. Hybridization of Alu probe to DNA on the filter was quantified by using the Betascope (Betagen 603, Betagen, Mountain View, CA). Topotecan-induced DNA-protein cross- linking was determined by taking the ratio of the aver- age counts of samples treated with topotecan to the counts from samples treated with vehicle. For in vivo testing, blood was collected from patients after the first dose of topotecan. The same procedure was employed to assess protein-DNA cross-links but the cells were not further exposed to the drug in vitro.

Results

Patient characteristics are outlined in Table 1. The me- dian age was 63 years (range, 25-71 years); 9 of the 12 (75%) were men. Eighty-three percent had a Zubrod performance status of 1 or lower. The median time from diagnosis to treatment with topotecan was 47 months (range, 22-96 months). All patients had received prior therapy with fludarabine; four (33%) were resistant to fludarabine. Four patients (33%) had also received ther- apy with chlorambucil; three of them were resistant to that agent (and fludarabine). At the time of treatment with topotecan, seven patients (58%) were Rai stage I- I1 and five (42%) were Rai stage 111-IV." The median

1106 CANCER March 2,1995, Volume 75, No. 5

Table 1. Patient Characteristics

Characteristics No. of uatients (%)

Age (yr) 260

Zubrod performance status 0-1

Rai stage 1-11 111-IV

Time from diagnosis (yr) 2 3

Prior fludarabine therapy Fludarabine resistant

Prior chlorambucil therapy ~

n = 12.

10 (83)

8 (67) 12 (100) 4 (33) 4 (33)

Table 3. Thrombocytopenia in Patients Receiving Topotecan: Platelet Counts (X103/pl)

Pretreatment Nadir End of course 1

237 222 335 163 14 112 161 52 173 155 93 158 145 28 188 143 54 294 123 25 145 118 3 145 56 12 58 47 8 16 22 5 33

hematologic parameters at the start of therapy were: leukocyte count 20 X 103/pl (range, 3 to 74 X 103/pl); absolute lymphocyte count 19.4 X 103/pl (range, 2.9 to 69 X 103/pl); hemoglobin level, 12 g/dl (range, 7.4 to 16 g/dl); and platelet count, 144 X 103/pl (range, 22 to 237 x 1 0 3 1 ~ 1 ) .

Response

All patients were evaluable for response. No patient achieved an objective response (95% confidence in- terval, 0-27%). One patient who began treatment with Rai stage IV disease had a decrease in lymph node size but did not show an increase in the platelet count; this patient received three courses of topotecan. Eight pa- tients were later treated with a fludarabine-based regi- men (fludarabine and Adriamycin or fludarabine and cisplatin). Five of these patients (63%) responded to subsequent treatment.

Toxicity

Nonhematologic toxicity is outlined in Table 2. The most common toxicity was nausea and vomiting, which was seen in six patients (50%). This was easily con-

trolled with the use of antiemetics. No toxicity higher than Grade 2 was noted.”

Four patients (33%) developed infections while re- ceiving topotecan. In three cases the infections were mi- nor: two patients developed bronchitis, and one patient developed sinusitis. All were treated with oral antibiot- ics as outpatients. The fourth patient was a 64-year-old woman with a history of chronic obstructive lung dis- ease, bronchiectasis, and recurrent pseudomonas lung infections. She developed a fungal pneumonia and died during the second course of topotecan. There was one other death on study, a 69-year-old man who died on the first course of therapy of an acute myocardial in- farction documented on postmortem exam.

All patients but one developed a decline in platelet counts (Table 3). Three patients started therapy with a platelet count < 100 x 103/pl (the patient who died early with a myocardial infarction is not shown). Among the eight patients who started with platelet counts > 100 X 103/pl, two (25%) developed thrombo- cytopenia < 50 X 103/pl and two (25%) had thrombo- cytopenia < 25 X 103/pl. The median duration of plate- let count < 50 X 103/pl was 9 days (range, 5-14 days). All patients recovered to their baseline platelet count within 28 days; there was no evidence of cumulative thrombocytopenia and no bleeding episodes were ob- served.

Topo-I-DNA Complex Measurements Table 2. Toxicity of Topotecan

No. of patients (%)

6 (50) 2 (17) 2 (17) 2 (17) 2 (17)

Figure 1 shows the results of the filter-binding assay using Mu-probe hybridization to quantify Top0 I-DNA com- plexes. The effects of exogenous topotecan (in vitro assay) were tested in nine patients. The effects of the topotecan dose administered in vivo were measured in eight pa- tients. All patients whose cells were tested in vitro showed an increase in cleavable complex formation after exposure

Toxicity (grade I 2)

Nausea/vomiting Diarrhea Rash Headache Alopecia -

Topotecan in CLLIOBrien e t al.

0 1

1107

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2m&m2 2PM

Discussion

H

G

F

E

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to topotecan, with interpatient heterogeneity. The levels of cleavable complex ranged from 2 to 7.5 times higher than in the control cells. Of the eight patients whose cells were also assessed for the formation of DNA-protein cross-linking after the first dose of topotecan, only two had a significant increase over the level before treatment. One patient had a 1.9-fold increase in cross-linking after the first dose of topotecan; the same patient showed the greatest cross-linking after topotecan in vitro with a 7.5- fold increase. Interestingly, the patient who showed the highest in vivo cross-linking after one dose of topotecan, a 2.9-fold increase over baseline, had a 2.8-fold increase measured in vitro.

Although CLL appears to be an attractive target for the use of drugs that interact with Topo-I, we noted no re- sponses in 12 CLL patients treated with topotecan. The development of transient, although significant, throm- bocytopenia suggests that this lack of response was not related to underdosing of the drug. All patients in this study had received prior treatment with fludarabine; however, only four patients were resistant to fludara- bine and the other patients had relapsed after therapy. The expected response rate to retreatment with fludar- abine in nonresistant patients would be 50%. Thus, this group of patients would not be highly resistant to sal- vage chemotherapy.

All patients evaluated showed an increase in Top0 I-DNA cleavable complex formation in cells exposed to topotecan in vitro. The heterogeneity of this increase was encouraging because there were interpatient differences in drug sensitivity that might correlate with clinical outcome. However, this could not be verified in this small number of patients who did not achieve remission. The lack of increase in the cleavable complex formation in cells exposed to topotecan in vivo may be due to the sensitivity level of the procedure. Plasma to- potecan levels after a 2 mg/m2 dose are approximately 100 nM; or 20-fold lower than the 2 yM used for the in vitro assay.I3 This suggests that topotecan levels higher than that achieved in vivo are required for the Topo-I directed activity to be observed.

Whether a continuous infusion schedule of topo- tecan over 5 days would be more effective in inducing such activity is questionable. Topotecan is cell-cycle specific; the percentage of dividing cells in patients with CLL is low, and protein-associated strand breaks are readily reversible after removal of topotecan. 14,15 Fol- lowing bolus administration of topotecan, the lactone form is rapidly hydrolyzed, and topotecan is quickly cleared from the plasma with a half-life of about 3 hours.I3 Burris et a1.16 examined the cytotoxicity of to- potecan in a human tumor colony-forming assay and found that prolonged exposure to topotecan yields a greater cell kill than if the same dose was given for only one hour. Thus, there is some rationale for prolonging the infusion time of this drug, especially in patients with CLL. In conclusion, topotecan administered as a 5-day bolus infusion produced no remissions in 12 patients with chronic lymphocytic leukemia.

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