Factors Affecting Topotecan Sensitivity in Human Leukemia Samples

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Factors Affecting Topotecan Sensitivity in Human Leukemia Samples"

SCOTT H. KAUFMANN,b.' STEVEN D. GORE,d LOUIS LETENDRE,' PHYLLIS A. SVINGEN ,' TIMOTHY KOTTKE,"

LOUISE B. GROCHOW," PHILIP J. BURKE,d ROSS C. DONEHOWER,d AND ERIC K. ROWINSKYd

'Department of Oncology "Division of Hematology, Department of' Medicine

M a y o Clinic Rochester, Minnesota 55905

"The Johns Hopkitis Oncology Center Baltimore, Maryland 21287

CHRISTOPHER A. BUCKWALTER,~ RICHARD J. JONES,~

INTRODUCTION

Preclinical studies have revealed a number of factors that can affect sensitivity of cells to camptothecin and its analogs.'-' Drug accumulation, content of the target enzyme topoisomerase I (topo I ) , susceptibility of top0 I to the inhibitory effects of these drugs, and the presence or absence of ongoing replication to con- vert drug-stabilized top0 I-DNA cleavage complexes into irreversible DNA double-strand breaks have all been shown to influence the cytotoxicity of camptothe- cins in model systems. Each of these factors is in turn a reflection of the balance between multiple cellular processes.

Relatively little is known about the cellular accumulation of camptothecin and its analogs. Several studies have demonstrated that the parent drug camptothecin is unaffected by P-glycoprotein and the multidrug resistance-associated protein (MRP), two broad-spectrum drug transporters (reviewed in refs. 1-4 and 6). In contrast, the cytotoxicity of topotecan, I0-hydroxy-7-ethylcamptothecin (SN-38). and 9-aminocamptothecin (9AC) is slightly diminished in cells that overexpress P- gly~oprotein.'-~ Conversely, it has recently been demonstrated that mitoxantrone- selected MCF-7/MX cells exhibit diminished topotecan accumulation and diminished sensitivity to several semisynthetic camptothecin analogs even though these cells do not overexpress P-glycoprotein or MRP.6 These latter observations raise

Support from the American Cancer Society (DHP-46) and National Institutes of Health (CA06973; U0I CA70095; U01 CA63437; U01 CA69912) is gratefully acknowledged. S. D. G. was the recipient of a Career Development Award from the American Cancer Society during the period of these studies. S. H. K. and R. J. J. are Scholars of the Leukemia Society of America. ' Address for correspondence: Scott Kaufmann. M.D., Ph.D., Division of Oncology Re-

search, Mayo Clinic, 200 First Street, S.W., Rochester. M N 55901; Tel: (507) 284-8950; Fax: (507) 284-3906; e-mail: Kaufmann.Scott@'Mayo.edu.

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KAUFMANN ef 01.: TOPOTECAN IN LEUKEMIA 129

the possibility that another transporter might affect the action of several camptothecin analogs currently in clinical trials.

The factors that affect top0 I expression and top0 I activity have been more extensively studied. Examination of elutriated chicken MSB-1 lymphoblasts revealed that top0 I content was relatively constant throughout the cell cycle in logarithmically growing cells.1° In addition, it has been reported that top0 I levels measured by activity assays or Western blotting do not change appreciably when normal hepatocytes or serum-deprived fibroblasts are stimulated to proliferate (reviewed in refs. 1 and 11). In contrast, transformation of murine fibroblasts with a variety of oncogenes results in modest elevations in top0 I activity.’* Likewise, transduction of v-rasH into NCI-H82 human small cell lung cancer cells is associated with increased top0 I activity.” Consistent with these results, examination of clinical tumor specimens has revealed that top0 I content is elevated in stage D colon cancer compared to normal colonic epithelium and in prostate cancer specimens compared to normal pro~ta te . ’~ .”

Selection of cell lines for camptothecin resistance also appears to affect top0 I content. Low-level resistance appears to be accompanied by decreases in top0 I content without other obvious changes in the top0 I enzyme.16-18 In contrast, selection for high-level resistance often results in the isolation of cells that have qualitative as well as quantitative changes in top0 I . In particular, highly resistant cells have been reported to express topo I species that are resistant to cleavage complex-stabilizing effects of the selecting agent and related camptothecin analogs as a result of mutations in the top0 1 gene (reviewed in refs. 4 and 5 ; see also refs. 19 and 20). To place this high level resistance in context, however, it is important to keep in mind the studies of Eng et a/ . ,2 ’ who demonstrated that cells selected for profound resistance to camptothecin in vivo (P388/CPT) displayed only an 8-fold elevation in camptothecin ICSO when compared to parental P388 cells in colony forming assays in vitro. One potential interpretation of these results is that relatively modest changes in ICsoare sufficient to change a curable neoplasm into one that will not respond to camptothecin analogs in vivo and that higher degrees of resistance (100- to 1000-fold) associated with top0 I mutations might be beyond the realm of what will be observed in the clinical setting.

Finally, the role of ongoing DNA synthesis in converting covalent top0 I-DNA complexes into cytotoxic damage has been extensively studied. Early investiga- tions (reviewed in ref. 1) revealed that camptothecin was much more toxic during S phase than during other stages of the cell cycle. Camptothecin treatment was shown to result in the formation of DNA double-strand breaks in the vicinity of DNA replication forks (reviewed in refs. 1, 4, 5, 2 2 , and 23). Treatment of cells with DNA synthesis inhibitors was shown to prevent the formation of the camptothecin-induced DNA double-strand breaks and the cytotoxicity of camptothecin analogs, strengthening the relationship between these two events. Collectively, these observations indicate that the cytotoxicity of camptothecin analogs is inti- mately tied to DNA replication.

The preceding preclinical studies provided a rationale for considering the possibility that differences in drug accumulation, top0 I content, or percentage of cells traversing the cell cycle might play a critical role in determining the sensitivity of human cancer cells to top0 I poisons. To assess this possibility, we examined

130 ANNALS NEW YORK ACADEMY OF SCIENCES

these parameters in conjunction with a series of clinical trials of topotecan in relapsedlrefractory acute leukemia and blast crisis chronic myelogenous leukemia (CML-BC).

TOP0 I CONTENT IN NORMAL LYMPHOHEMATOPOIETIC CELLS

To provide a background for examination of topo I content in leukemia cells, we first examined the possibility that top0 I expression might change during myeloid maturation." Treatment of HL-60 human myelomonocytic leukemia cells with dimethylsulfoxide or retinoic acid in vitra results in maturation of these cells toward granulocytes (reviewed in ref. 24). This maturation is accompanied by a 5- to to-fold decrease in cellular top0 1 content (FIG. ]A). Interestingly, this decrease in top0 I content occurred without any change in top0 I mRNA levels on Northern blots," raising the possibility that top0 I mRNA level might not be a good indicator of cellular topo I polypeptide content. The decrease in top0 I polypeptide content in the maturing HL-60 cells was preceded by a change in the staining pattern within nuclei. In logarithmically growing HL-60 cells, top0 1 was localized to the nucleus but was concentrated in nucleoli (nrrolzv, FIG. 1C). In contrast, top0 I was found throughout nuclei without prominent nucleolar accentuation in the maturing cells (FIG. 1D).

These changes were not limited to the HL-60 cell line. Western blotting using highly enriched cell fractions revealed that granulocytic maturation in vivo was

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FIGURE 1. Alterations in top0 I content and localization during granulocytic maturation of HL-60 cells in vifro. A & B, Western blotting. HL-60 cells treated with 1.3% (vlv) DMSO for 1-8 days (lanes 5-12. respectively) were sedimented on Ficoll-Hypaque gradients to remove dead cells and prepared for SDS-polyacrylamide gel electrophoresis. Lanes contained material from 3 x 10' DMSO-treated cells ( h i e s 5-12) or 3 x lo5 (lanes Z), 1.5 x los ( / a n e s 2 ) , 0.75 X 1O5(/unes3) and 0.3 x lo5 ( l a n e 4 untreated cells. Afterelectrophoresis and transfer to nitrocellulose, samples were probed with the C-21 monoclonal anti-top0 I (A) or an antibody against histone HI (B). C-E, immunoperoxidase staining for top0 I. Samples of HL-60 cells treated with DMSO for 0 (C), 4 (D) or 7 days (E) were stained for top0 I as described." Corresponding fields were photographed under bright field (C-E) or phase contrast (C'-E') illumination.

KAUFMANN et al.: TOPOTECAN IN LEUKEMIA 131

FIGURE 2. Top0 I staining of normal marrow cells. A marrow fraction enriched in leuko- cytes was stained with C-21 monoclonal anti-top0 I followed by peroxidase-labeled anti- mouse IgM using previously published techniques." The same field was examined by bright field (left pane l ) and phase contrast microscopy (right pane l ) . Immature cells (e.g., cell # I ) contain phase-dense nucleoli (arrow) that stain more intensely for top0 I than the remainder of the nucleus. As the cells progressively mature along the myeloid lineage (cells #2-4, respectively), the nucleoli and nucleolar accentuation of top0 I staining become difficult to discern. Normal granulocytes retain a signal for top0 I (cell #4) but have much less nuclear volume than less mature cells (e.g.. cell # I ) , presumably accounting for the decreased amount of top0 I observed when mature granulocytes are examined by Western blotting.lI

also accompanied by a 10-fold decrease in top0 I content." Immunohistochemical staining revealed that top0 I was detected in every nucleated cell in the marrow (FIG. 2). As was the case in HL-60 cells (FIG. I ) , nucleoli became difficult to identify and nucleolar accumulation of top0 I became difficult to discern as cells matured along the myeloid pathway (FIG. 2). Conversely, entry of purified T lymphocytes into the cell cycle after stimulation with mitogens was accompanied by a threefold increase in top0 I content and a marked accumulation of the enzyme in the nucleolus." These observations suggest that top0 I content and localization will vary depending on the degree of maturation of the lymphohematopoietic cells being examined.

A CLINICAL TRIAL OF SINGLE-AGENT TOPOTECAN IN RELAPSED/REFRACTORY LEUKEMIA

Based on the observation that granulocytopenia was the dose-limiting toxicity of topotecan in solid tumor patients (reviewed in refs. 1,2), we performed a series of trials of topotecan in acute leukemia and CML-BC. Clinical and laboratory results from the first of these trials are summarized below. These results illustrate the difficulty in translating the preclinical observations on drug sensitivity described above (see INTRODUCTION) into tests that have clinical predictive value.

Two phase I trials have examined the toxicity of a five day continuous infusion of topotecan administered every 2 1 days to patients with refractorylrelapsed leukemia and CML-BC.'5.26 In the Johns Hopkins trial, 17 patients received 33 cycles of therapy at doses ranging from 0.7 mg/m2/d to 2.7 mg/m2/d. The maximum toler-

I32 ANNALS NEW YORK ACADEMY OF SCIENCES

ated dose (MTD) was found to be 2.1 mg/m2/d (compared to 0.67 mg/m2/d in solid tumor patients”). When the MTD was exceeded, severe oral and penanal mucositis was observed in all patients.

In conjunction with this phase I trial, steady-state serum concentrations of topotecan were measured by high performance liquid chromatography. Mean steady-state concentrations of topotecan (lactone + carboxylate) ranged from 3 to 72 nM in these patients, with a mean steady-state concentration of 25.9 * 6.7 nM at the MTD.

Some degree of antileukemic effect was observed during all courses administered to patients with circulating blasts. Complete clearance of blasts circulating in the blood stream was observed in 0/4 courses at 0.7-0.9 mg/m2/d, 1/6 courses at 1.2 mg/m’/d, 2/6 courses at 1.6 mgim’ld, and 8/15 courses at 2 2 . 1 mg/m’/d. In addition, a brief complete response (defined as 4% blasts in the marrow and reconstitution of normal hematopoiesis) was observed in a patient with CML-BC who received 2.1 mg/m’/d topotecan for two courses.

EXAMINATION OF FACTORS AFFECTING TOPOTECAN SENSITIVITY

As an adjunct to this clinical trial, several aspects of top0 I-mediated drug action were examined in )*;fro. For these studies, bone marrow aspirates harvested immediately prior to the institution of topotecan therapy were fractionated on Ficoll-Hypaque gradients to enrich the leukemic cell populations. The ability of topotecan to inhibit leukemic cell colony formation (CFU-L) was examined by plating T-cell depleted leukemia cells in 1.32% (w/v) methylcellulose containing 0-50 nM topotecan and evaluating colony formation 3-6 days later. This continuous exposure paradigm was chosen to mimic the clinical drug treatment protocol. Results of these experiments (FIG. 3B) were compared to similar cloning experiments that examined topotecan sensitivity of myeloid colony forming cells from normal bone marrows (CFU-GM, FIG. 3A).

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FIGURE 3. Sensitivity of normal CFU-GM (A) and leukemic CFU-L (B) to topotecan in vitro. T-cell depleted normal (A) or leukemic marrow mononuclear cells (B) were plated in 1.32% (w/v) methylcellulose and exposed to the indicated concentration of topotecan (lactone + carboxylate) continuously until colony formation was assessed.

KAUFMANN el al. : TOPOTECAN IN LEUKEMIA 133

Several aspects of these cloning studies are noteworthy. First, CFU-L were successfully cultured from only five of eleven leukemic marrows on which this assay was performed. This result is consistent with the difficulty that other con- temporary studies noted in culturing CFU-L in vitro. Second, the cloning efficiency of the CFU-L ranged from 0.008% to 1.7%. Stated another way, even though the original marrows contained >80% blasts, only 0.008-1.7% of these malignant cells gave rise to colonies in vitro. Because the cloning efficiency is so low, it is not clear that response of these cells will be representative of all of the leukemic cells. Moreover, it is not clear that parameters like drug accumulation, cell cycle distribution, and top0 I content measured in the leukemia specimen as a whole will be representative of the same parameters in the clonogenic leukemia cells.

In these clonogenic assays, the L D ~ o for TPT in the CFU-L ranged from 6 nM to 22 nM (FIG. 3B), a range that is similar to the LD90 of the CFU-GM in normal marrows (FIG. 3A). Assuming that relatively little topotecan is protein bound and that the equilibrium between topotecan lactone and the open ring carboxylate is similar at pH 7.4 in vitro and in vivo, comparisons of the LD90 observed in vitro and the steady state concentration achieved in vivo should be valid. Overall, the range of steady-state serum concentrations achieved at the MTD of this agent (25.9 t 6.7 nM) exceeded the range of LDms observed in CFU-L in vitro (14 t 8 nM). On the one hand, this observation probably accounts for the antileukemic effects (decreased circulating blasts) in these patients. On the other hand, this observation gives rise to concern that the steady-state topotecan concentrations in most patients will be fairly close (within a factor of two or three) to the LD90 of their clonogenic leukemia cells, making it difficult to envision how one would achieve the multiple logs of cell kill that are probably required for eradication of the leukemia.21

Drug accumulation, top0 I content, and cell cycle distributions were examined in vitro in the same leukemia samples. Topotecan accumulation was measured by flow microfluorimetry.8 To permit comparison of samples exposed to drug and analyzed at different times over the two year course of this study, cellular topotecan content was expressed as a ratio of topotecan fluorescence in the clinical specimen to topotecan fluorescence in logarithmically growing KG la human myeloid leukemia cells subjected to the same assay on the same day. Results of these assays (FIG. 4) indicated that cellular topotecan accumulation varied over a two- fold range. In the single patient whose cells were examined at the start of treatment and six weeks later at recurrence, there was no decrease in topotecan accumulation over time (data not shown).

Top0 I content was examined by Western blotting using methods depicted in FIGURE 1A. To correct for differences in sample loading, the same blot was probed with an antibody that recognizes histone H1, a nuclear protein that is present in identical amounts in all diploid cells (FIG. 1B). After normalization for sample loading, top0 I levels in the leukemia samples were compared to levels in logarithmically growing HL-60 cells (which were arbitrarily assigned a value of 1 .O). Samples containing >80% blasts were available from 13 of the 17 patients enrolled in this study. Top0 I content in these samples varied over a 10-fold range, from 8% of the top0 I content in an equal number of HL-60 cells to 85% of the top0 I

134

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FIGURE 4. Relative steady-state topotecan a t - cumulation in leukemic blasts. Blasts or KGla cells were incubated for 30 min at 37°C with 25 p M topotecan, then subjected to flow microfluorimetry.* Data were plotted as the ratio of the mean fluorescence of the clinical specimen to the mean fluorescence of the KGla cells treated simultaneously.

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content in HL-60 cells (FIG. 5A). Interestingly, top0 I content varied according to the type of leukemia, being relatively low in CML-BC (including the patient who achieved a complete remission on this trial), intermediate in acute myelogenous leukemia, and somewhat higher in acute lymphocytic leukemia.

Although the conclusions are undoubtedly limited by the small sample size and the heterogeneity of the leukemia patients enrolled, there was no correlation between top0 I content and response to therapy (FIG. 5A), no correlation between topo I content and percentage of cycling cells as assessed by flow cytometry after propidium iodide staining (FIG. 5B). and no correlation between top0 I content and sensitivity to topotecan in the clonogenic assays (FIG. 5C). These results were disappointing but not altogether unexpected. Although it has been shown in carefully controlled model systems that decreases in top0 I content are associated with resistance to top0 I-directed agents and increases in top0 I content are associated with hypersensitivity to top0 I-directed agents (see, for example, refs. 28 and 29). the converse has not been shown. Indeed, when cell lines from unrelated individuals bearing the same tumor type have been examined in vitro, it has been difficult to demonstrate any relationship between top0 I content and sensitivity to camptothecin analog^.^'-^* In addition, the low cloning efficiency of CFU-L (see above) makes it difficult to evaluate whether the top0 I content measured in a leukemic marrow specimen containing S O % blasts is truly representative of the clonogenic cells that proliferate in vitro and in the patient.

In summary, these studies illustrate the difficulty in studying sensitivity to the top0 I poisons in clinical material. On the one hand, the recent phase I and phase I1 trials probably represent the only time that it will be possible to compare the response of patients receiving single-agent top0 I poisons to results of assays performed in vitro. On the other hand, the phase I setting is a difficult one for assessing correlations because patients with many different types of neoplasms ( e . g . . multiple types of aggressive leukemia) that receive a variety of drug doses. Even though the doses are more uniform in the phase I1 setting, the heterogeneity of the neoplastic cells in terms of degree of maturation and proliferative status (to say nothing of contamination by normal cells) is likely to make it difficult to ascertain any relationship between top0 I content and response of clonogenic cells.

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FIGURE 5. Relationship between relative top0 I content (determined by Western blotting as described in the text)26 and leukemic phenotype (A), % GZ + S phase cells in the marrow sample (B) or sensitivity of CFU-L to topotecan in vifro (C). Top0 1 levels are expressed relative to an equal number of HL-60 cells. In panel A, top0 I levels in marrow mononuclear cells from 5 normal controls are shown for comparison. The solid circle represents the sample from the single patient who achieved a complete remission on this trial. Data from a marrow containing only 30% blasts has been omitted from this analysis, accounting for the difference in sample numbers between Figure 3B and panel C.


DEVELOPMENT OF NEW ASSAYS OF DRUG SENSITIVITY

The low cloning efficiency of the CFU-L in vitro (see above), coupled with the question of whether the response of the CFU-L is representative of the tumor population as a whole, prompted us to attempt to devise other techniques for measuring the effect of camptothecin analogs on human leukemia cells. Two of these assays are briefly illustrated.

First, based on the observation that the nuclear localization of top0 I appears to be altered during the course of granulocytic maturation in vitro (FIG. IC,D) and in vivo (FIG. 2 ) , we examined the acute effect of inhibiting DNA or RNA synthesis on the subnuclear localization of top0 I. Results of these studies are shown in FIGURE 6. In untreated K562 leukemia cells, top0 I is localized throughout the nuclei but is concentrated in nucleoli (arrows, FIG. 6A’). After treatment with the DNA polymerase inhibitor aphidicolin at concentrations that inhibit DNA synthesis by >95%, there was no change in the subnuclear localization of top0 I.33 In contrast, treatment with the RNA synthesis inhibitor 5,6-dichloro-P-l-D- ribofuranosylbenzimidazole (DRB) at concentrations that inhibited RNA synthesis by >70% resulted in an altered pattern of top0 I fluorescence, with a decrease in the nucleolar signal (FIG. 6B’). These changes occurred without any detectable alteration in cellular top0 I content or top0 I Further studies indicated that camptothecin and topotecan also induced an apparent relocalization of top0 I to non-nucleolar portions of the nucleus. Interestingly, when camptothecin-resistant DC3FIC-10 Chinese hamster lung cells were compared to parental cells, the increase in topotecan concentration required to kill the DC3F/C-10 cells (FIG. 6D) was reflected by an increase in the concentration of topotecan required to cause the translocation of top0 I to non-nucleolar portions of the nucleus (FIG. 6C). These observations raise the possibility that it might be possible to screen for high-level topotecan resistance in a population of tumor cells by exposing the cells to increasing concentrations of topotecan in vitro and subsequently examining the subnuclear distribution of top0 I by histochemistry. Whether this assay will have any utility when applied to clinical leukemia specimens remains to be determined.

Based on the demonstration that camptothecin analogs appear to kill HL-60 cells by inducing a p o p t ~ s i s , ~ ~ - ~ ~ we have also begun to assess the possibility that measurement of topotecan-induced apoptosis might provide a means of distin- guishing between leukemia samples from patients with sensitive and resistant dis- ease. Methods for measuring apoptosis in clinical samples (and in tissue culture) are currently evolving rapidly. Because of concern that terminal transferase-based methods might detect necrotic cells as well as apoptotic cells,37 we elected not to use a terminal transferase-based method. Likewise, because each intact cell might give rise to multiple subdiploid apoptotic bodies ( e . g . , FIG. 9 in ref. 38), we elected not to utilize flow cytometry-based methods. Instead, we have utilized classical agarose gel electrophoresis to search for the internucleosomal DNA frag- mentation that is characteristic of apoptotic cell death. Results of one of these assays are shown in FIGURE 7. Treatment of leukemic blasts for 24 h with a high topotecan concentration in vitro was associated with induction of internucleoso- ma1 DNA cleavage, whereas no cleavage was observed in diluent-treated cells. Whether this type of assay will have any predictive value is currently unknown.

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FIGURE 6. Effect of RNA synthesis inhibitors on top0 I accumulation in nucleoli. A&B, Effect of 30 pM DRB on top0 1 signal in KS62 cells. KS62 cells were incubated with diluent (A, A') or 30 y M DRB (B, B') for 60 min, fixed. and stained with C-21 anti-top0 1 followed by rhodamine- conjugated anti-mouse IgM, and examined by phase contrast (A, B) or fluorescence microscopy (A', B'). In control cells there is a strong signal for top0 I in nucleoli (arrow in A, A'). After treatment with DRB, the signal for top0 I in nucleoli is diminished (B') even though nucleoli are readily visible by phase contrast (B) and electron C, Effect of of various concentrations of topotecan on the % of cells displaying a topo I signal in nucleoli: -, DC3F hamster lung fibroblasts; -------, camptothecin-selected DC3F/C-IO cells. D, clonogenic survival of DC3F (-) and DC3F/C-10 (-------) cells in the presence of various concentrations of topotecan.

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FIGURE 7. Induction of apoptosis by topotecan in leukemic blasts in virro. Marrow mononuclear cells (>99% myeloblasts) from a patient with relapsed acute myelogenous leukemia were incubated for 24 h at 37°C in RPMI 1640 medium containing 20% ( v h ) autologous patient serum in the presence of 10 pM ( lane 3) , I p M (lane 4, 0.3 pM (lane 5 ) or 0.1 pM (lane 6 ) topotecan or in diluent (0.1% DMSO, lane 2 ) . At the completion of the incubation, cells were sedimented and prepared for agarose gel e l ec t rophore~ i s .~~ Samples containing DNA from 1.25 x lo5 cells were applied to each lane. Lane 1, A Hind111 fragments used as molecular weight markers.

EVALUATION OF TOPOTECAN-CONTAINING COMBINATIONS IN HUMAN LEUKEMIA CELL LINES

The observation that the LDws of clonogenic leukemia cells in virro are close to the steady-state serum concentrations of topotecan in v ivo suggests that single- agent topotecan is unlikely to have overwhelming clinical activity against acute leukemias. The low response rates observed in phase I trial^",^^,)^ are consistent with this view. This pessimism, however, must also be tempered with the realiza- tion that patients enrolled in these phase I trials had previously received extensive treatment with other antileukemic agents. The fact that clearance of circulating blasts was observed in a substantial fraction of patients (see above) raises the possibility that topotecan might be useful when combined with other agents that also have antileukemic effects. Toward this end, we have examined the interaction between topotecan and several antineoplastic agents in human leukemia cell lines in vitro.

The methods for examining the interaction between two agents are currently a matter of considerable debate.40-42 For these studies, we examined the cytotoxicity of topotecan alone, a second agent alone, and a fixed ratio of topotecan plus the second agent. Results obtained with several of these combinations are illustrated in FIGURE 8. When HL-60 cells were treated with topotecan + cytarabine, the cytotoxicity of the combination was similar to the cytotoxicity of topotecan alone


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FIGURE 8. Interaction of topotecan with other antineoplastic agents in human leukemia cell lines. A,B, HL-60 cells were treated with topotecan (TPT) alone, cytarabine alone, or a fixed 1 :6.7 molar ratio of topotecan and cytarabine. After a 24 h incubation at 37"C, cells were washed and plated in drug-free 0.3% agar as de- ~c r ibed .~ ' Colonies were counted 10-14 days later. C,D, HL-60 cells were plated in 0.3% agar containing topotecan, carboplatin (CBDCA), or a fixed 1 : 1000 molar ratio of topotecan and carboplatin. Colonies were counted 10-14 days later. E,F, K562 cells were plated in 0.3% agar containing topotecan, carboplatin, or a fixed I : 1000 ratio of topotecan and carboplatin. G , K562 cells were plated in 0.3% agar containing topotecan, chlorambucil (not shown), or a fixed I :500 ratio of topotecan and chlorambucil. H, KS62 cells were plated in 0.3% agar containing topotecan, 4-hy- droperox ycyclophosphamide (4HC. not shown) or a fixed I : 167 ratio of topotecan and 4-hydroperoxycyclophospha- mide. In each case, the ratio of the two agents was approxi- mately the ratio of the individ- ual ICSO values determined in previous experiments. Control plates in each experiment contained 250-500 colonies. Ver- tical bars, mean & standard deviation of quadruplicate ali- quots.

(FIG. 8A). Additional experiments in other cell lines using other exposure proto- cols have likewise led to the conclusion that the interaction between topotecan and cytarabine is less than additive.43 In contrast, treatment of the same cell line with topotecan + carboplatin led to a marked increase in the cytotoxicity compared to either drug alone (FIG. 8C.D). A similar increase in cytotoxicity was observed when topotecan + carboplatin were used to treat a second human leuke-


mia line (FIG. 8E,F) and when carboplatin was replaced with the alkylating agent chlorambucil (FIG. 8G). Interestingly, the alkylating agent 4-hydroperoxycyclo- phosphamide did not have this effect (FIG. 8H). These results, coupled with additional results in other human tissue culture cell lines,43 have led to the suggestion that top0 I poisons might be more active when combined with certain DNA- damaging agents rather than with antimetabolites. Additional preclinical and clinical studies will be required to assess this possibility.

PROSPECTS FOR THE FUTURE

Studies reviewed elsewhere in this volume indicate that the camptothecin analogs are likely to be important new additions to the medical oncologist’s therapeu- tic armamenterium. In particular, irinotecan has shown unique clinical activity against non-small cell lung cancer and fluoropyrimidine-resistant colon cancer. Promising response rates have also been observed in phase I1 trials of this agent in small cell lung cancer as well as carcinomas of the pancreas, stomach, ovary, cervix and breast. Topotecan likewise has demonstrated activity in phase I1 trials in carcinomas of the head and neck, ovary, and breast as well as small cell lung cancer.

Biological assays that accurately assess the probability of a particular neoplasm’s response to this class of agents would be extremely useful in the clinical setting. Based on the existing understanding of the mechanism of action of this class of agents, we examined a series of parameters that were originally considered potential predictors of response. Even in a relatively ideal setting ( i . e . , a readily accessible neoplasm with low contamination by normal cells), we were unable to discern clear-cut relationships between pretreatment characteristics of the neoplastic population and response to treatment in vitro or in vivo. A second genera- tion of assays has been designed to readdress this issue. Whether these assays will have more predictive value is currently under investigation.

Whether or not predictive assays are devised, it is highly likely that the clinical development of top0 I poisons will continue. Phase I and phase I1 trials of top0 I poisons in combination with other agents are currently ongoing. The preclinical studies presented above suggest that the interactions between topotecan and some drugs will be less than additive, whereas the interactions between topotecan and other drugs will be additive or even synergistic. It remains to be determined whether the additive or synergistic interactions will be deleterious (leading to more toxicity in normal tissues) or beneficial in the clinical setting.

ACKNOWLEDGMENTS

We gratefully acknowledge collaborations with Alex Adjei, David Peereboom, Yung-Chi Cheng, Martin Charron, Amy Lin, Akihiko Tanizawa, Yves Pommier, and Judith E. Karp that contributed to the studies reviewed in this paper. These studies were made possible by the skilled care of the nurses, physician’s assistants, and house officers on the Adult Leukemia Service of the Johns Hopkins Oncology Center.


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