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Cancer Therapy: Clinical |
Authors' Affiliations: 1 Division of Hematology, Department of Medicine; Divisions of 2 Oncology Research and 3 Medical Oncology, Department of Oncology; and 4 Department of Biostatistics, Mayo Clinic College of Medicine, Rochester, Minnesota and 5 Greenebaum Cancer Center, University of Maryland, Baltimore, Maryland
Requests for reprints: Scott Kaufmann, Mayo Clinic, 1342 Guggenheim, 200 First Street Southwest, Rochester, MN 55901. Phone: 507-284-8950; Fax: 507-284-3906; E-mail: kaufmann.scott{at}mayo.edu.
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Abstract |
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Experimental Design: Patients received topotecan and carboplatin by 5-day continuous infusion at nine dose levels. Patients achieving a complete remission received up to two additional courses for consolidation. Plasma topotecan and ultrafilterable platinum were assayed on days 1 to 5. In addition, pretreatment levels of various polypeptides in leukemic cells were examined by immunoblotting to assess possible correlations with response.
Results: Fifty-one patients received a total of 69 courses of therapy. Dose-limiting toxicity consisted of grade 4/5 typhlitis and grade 3/4 mucositis after one course of therapy or grade 4 neutropenia and thrombocytopenia lasting >50 days when a second course was administered on day 21. Among 45 evaluable patients, there were 7 complete remissions, 2 partial remissions, 1 incomplete complete remission, and 1 reversion to chronic-phase chronic myelogenous leukemia. Topotecan steady-state plasma concentrations increased with dose. No accumulation of topotecan or ultrafilterable platinum occurred between days 1 and 5 of therapy. Leukemic cell levels of topoisomerase I, checkpoint kinase 1, checkpoint kinase 2, and Mcl-1 correlated with proliferating cell nuclear antigen but not with response. In contrast, low Bcl-2 expression correlated with response (P = 0.014, Mann-Whitney U test).
Conclusions: The maximum tolerated dose was 1.6 mg/m2/d topotecan plus 150 mg/m2/d carboplatin. The complete remission rate in a heavily pretreated population was 16% (33% at the highest three dose levels). Responses seem to correlate with low pretreatment blast cell Bcl-2 expression.
Previous trials demonstrated schedule-dependent activity of carboplatin in patients with relapsed and refractory acute leukemia. Although this agent did not induce remissions when administered as a daily bolus for 5 consecutive days (7), carboplatin was active against relapsed acute leukemias when administered as a 5-day continuous infusion (8–10). Complete remission (CR) rates of 16% to 36% (9, 10) were observed in phase II trials, and most CRs in the phase I trial of carboplatin occurred at doses below 200 mg/m2/d (8). The dose-limiting toxicity (DLT) of carboplatin in leukemia patients was prolonged neutropenia. Other toxicities included renal failure, particularly in the setting of nephrotoxic antibiotics and amphotericin (8, 9).
Topotecan, a water-soluble semisynthetic topoisomerase I poison, has also been investigated in relapsed and refractory acute leukemia. Although this agent had little antileukemic activity when administered as a daily bolus for 5 days (11), CRs were observed in three separate phase I trials that administered topotecan by 5-day continuous infusion (12–14). Oral and perianal mucositis were the DLTs of topotecan administered to adult patients on this schedule (12, 13). Topotecan also exhibited activity in myelodysplastic syndrome as a single agent and in combination with cytarabine (15). There remains considerable interest in building on these results, as indicated by a large number of phase I and phase II trials of various topotecan-containing combinations over the past several years (reviewed in ref. 11; see also refs. 16–18).
Laboratory investigations have revealed that topotecan and cisplatin exhibit synergy when administered simultaneously to a variety of cell lines in vitro (19–21) or to tumor-bearing mice in vivo (22). Likewise, topotecan and carboplatin synergize in human leukemia cell lines in vitro (23). The mechanism of this synergy is not completely understood, but recent studies suggest that platination of DNA might enhance trapping of topoisomerase I-DNA covalent complexes, thereby contributing to enhanced toxicity of the combination (24, 25).
Preclinical studies have also identified cellular properties that might affect topotecan sensitivity (reviewed in refs. 26–28). Early studies showed that cells with higher topoisomerase I polypeptide levels are more sensitive to topoisomerase I poisons. When topoisomerase I content was examined in samples of relapsed or refractory leukemia that contained at least 80% blasts, however, topoisomerase I content did not correlate with response to topotecan in either phase I (13) or phase II (29) trials. More recent reports have identified several additional factors that could potentially affect topotecan sensitivity. First, topotecan uptake is reduced in cells that overexpress the ATP-binding cassette transporter breast cancer resistance protein (BCRP; refs. 28, 30). Importantly, BCRP has been detected in leukemic blasts, although a correlation between BCRP expression and poor response to therapy has not been consistently observed (30, 31). Second, levels or activity of DNA damage–activated kinases, particularly ataxia telangiectasia mutated- and Rad3-related (ATR) protein and checkpoint kinase 1 (Chk1), affect sensitivity to topoisomerase I poisons (reviewed in refs. 28, 32). Third, the ability of cells to degrade drug-stabilized covalent topoisomerase I-DNA complexes correlates with resistance to topoisomerase I poisons (28, 33). Finally, studies dating back over a decade have suggested that cells expressing elevated levels of the antiapoptotic protein Bcl-2 become resistant to camptothecin analogues (26, 27, 34), although the importance of this mechanism in the clinical setting remains incompletely explored.
Based on the preclinical data showing synergy between topotecan and platinating agents, the observed single-agent activity of carboplatin and topotecan in refractory and relapsed acute leukemia, and the nonoverlapping nonhematologic toxicities of these agents, we performed a phase I and pharmacologic study of topotecan and carboplatin in patients with relapsed/refractory acute leukemia and AP-CML or BC-CML. The goals of this study were to (a) determine the maximum tolerated dose (MTD) of the two agents administered simultaneously as a 5-day continuous infusion, (b) describe the DLTs of the combination, (c) seek preliminary evidence of activity of this regimen, (d) assess the effect of carboplatin on steady-state topotecan levels, and (e) correlate responses with expression of polypeptides implicated in topotecan resistance.
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Patients and Methods |
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18 years) with AML or ALL who failed to respond to intensive conventional chemotherapy or relapsed after a CR were eligible for this study. Acute leukemia patients were generally allowed 
2 prior induction regimens. Patients with AP-CML and BC-CML were entered before imatinib mesylate became available and were eligible without prior chemotherapy or after one prior induction regimen.
Additional eligibility criteria included Eastern Cooperative Oncology Group performance status 0 to 2 at the time of treatment, adequate hepatic (total bilirubin, <2 mg/dL) and renal (creatinine, 1.5 mg/dL) function, and recovery from significant toxicities of preceding therapy. Patients receiving hydroxyurea or glucocorticoids to prevent leukostasis were eligible if these agents were stopped 24 hours before initiation of therapy. Patients were ineligible if they had evidence of active central nervous system leukemia, uncontrolled infection at the time of enrollment, or, because of concern about the renal toxicity of concurrent carboplatin (8, 9), aminoglycoside or amphotericin treatment in the preceding 7 days. The study was approved by institutional review boards of the participating institutions in accordance with the policies of the Department of Health and Human Services, and written informed consent was obtained from each patient before chemotherapy.
Patient evaluation. Before enrollment, a history and physical examination were completed along with the following laboratory studies: complete blood count, prothrombin time, activated partial thromboplastin time, fibrinogen, serum creatinine, sodium, potassium, uric acid, calcium, phosphate, magnesium, total protein, albumin, total bilirubin, direct bilirubin, aspartate aminotransferase, and alkaline phosphatase. The physical examination and chemistry tests were repeated twice weekly during each cycle. Complete blood counts were obtained daily until the absolute neutrophil count and platelet count were >500/mm3 and 20,000/mm3, respectively.
Drug formulation and administration. Topotecan supplied by the Division of Cancer Treatment, National Cancer Institute (Bethesda, MD) as formulation AC/AF in vials containing a lyophilized mixture of 5 mg topotecan (pH adjusted to 3), 60 mg mannitol, and 25 mg tartaric acid was reconstituted in 2 mL sterile water. Carboplatin (Bristol Myers Squibb, Princeton, NJ) was reconstituted in sterile water at 10 mg/mL. Based on studies showing compatibility of the two drugs, topotecan and carboplatin were mixed in 5% dextrose such that one third of the daily dose was administered in 500 mL over 8 hours. Both agents were infused continuously through a central venous catheter for 120 hours.
Initial dosage and escalation scheme. The starting doses of 0.5 mg/m2/d topotecan and 150 mg/m2/d carboplatin by 5-day continuous infusion, which represent one fourth and one half the respective single-agent MTDs determined in previous phase I studies (8, 12, 13), were chosen in consultation with National Cancer Institute staff because of anticipated synergistic effects of the two agents. The initial trial design called for escalation of carboplatin to 200 mg/m2/d and topotecan to 0.75, 1.0, 1.3, 1.6, and 2 mg/m2/d. When prolonged myelosuppression (a DLT of carboplatin) was observed at the first dose level, carboplatin was fixed at 150 mg/m2/d and topotecan was increased to 1.25 mg/m2/d before escalating the carboplatin. After DLT was observed at 1.30 mg/m2/d topotecan and 200 mg/m2/d carboplatin, carboplatin was de-escalated to 150 mg/m2/d and topotecan was again increased.
Ancillary treatment. All patients received 300 mg/d allopurinol on days 1 to 7 of induction therapy and 400 mg p.o. or 5 mg/kg i.v. q8h acyclovir on days 8 to 28 of each cycle. After a thrombosed hemorrhoid and a perirectal tear were observed in patients straining at stool on days 21 to 28, patients received daily bulk laxatives (psyllium) and a stool softener (docusate sodium) unless diarrhea was present. Patients with neutropenic fevers received empirical antibiotics with anti-pseudomonal coverage. Aminoglycosides and amphotericin B were avoided while carboplatin and topotecan were infusing.
Retreatment and consolidation therapy. Patients who did not achieve a CR after one course of therapy were initially eligible for a second course on day 21 or later if the blast index (% cellularity x % blasts) was 25% of the pretreatment value. When three patients at dose level 1 had cytopenias lasting >50 days after retreatment on day 21, no further patients received a second induction course. Patients in CR received up to two courses of consolidation therapy that consisted of topotecan plus carboplatin administered by 5-day continuous infusion every 6 weeks beginning on days 60 to 70. Dose reductions of one dose level were permitted for DLT.
Definition of dose-limiting toxicity and maximum tolerated dose. Drug-related adverse events were graded by the National Cancer Institute Common Toxicity Criteria version 1.0. DLT was initially defined as (a) grade 3 nonhematologic toxicity or (b) grade 4 hematologic toxicity persisting beyond day 28. When the first six patients had grade 4 neutropenia and thrombocytopenia beyond day 28 but tolerated this well because of relatively mild nonhematologic toxicity, the protocol was amended to define grade 4 hematologic toxicity persisting beyond day 35 as a DLT.
Three patients without prior stem cell transplant (SCT) were to be enrolled at each dose level. If one of three experienced DLT, up to three additional patients were to be entered at that dose level. If two patients experienced DLT on the first cycle, that dose level was considered too toxic; and the previous level at which zero or one of six patients experienced DLT was defined as the MTD.
Out of concern that prior SCT might alter tolerance to this regimen, a separate dose escalation involving one to two patients per level was conducted to determine the MTD for these patients. Because of slow accrual, this arm was terminated when the non-SCT arm was completed. Toxicities and response for the SCT patients are reported separately, but pharmacokinetics and ancillary studies are combined.
Evaluation of response. To evaluate response, bone marrow aspirates and biopsies were obtained within 48 hours before therapy and again on day 10 to 14 after initiation of therapy. If the day 10 to 14 marrow showed no evidence of disease, the marrow was repeated on day 28 and again after peripheral counts recovered. If the day 10 to 14 marrow showed residual leukemia, a second marrow was initially performed on day 20 to 22 to determine eligibility for a second induction course.
As suggested by the National Cancer Institute–sponsored Workshop on Definitions of Diagnosis and Response in Acute Myeloid Leukemias (35), a CR for patients with AML or ALL was defined as a cellular marrow (cellularity, >20%) with <5% blasts and with adequate peripheral counts (absolute neutrophil count, >1,500/µL; platelets, >100,000/µL). For AP-CML, a morphologic CR was defined as <5% blasts in the marrow and with adequate peripheral counts (absolute neutrophil count, >1,500; platelets, >100,000). Karyotypes were repeated on AP-CML patients who achieved a morphologic CR.
Pharmacokinetic analysis. Blood samples (2 x 7 mL) were drawn into heparinized tubes before and at 24, 48, 72, 96, and 120 hours into the infusion. Blood was immediately cooled on ice and sedimented at 800 x g for 10 minutes at 4°C. Aliquots of plasma for topotecan determination were frozen at –70°C. Four aliquots (500 µL each) of unfrozen plasma were sedimented at 2,000 x g for 60 minutes at 4°C in Aminco Centri-free micropartition devices. These ultrafiltrates were frozen at –70°C until platinum content was measured.
Total topotecan was assayed as described by Beijnen et al. (36). To convert topotecan carboxylate to lactone, 10 µL H3PO4 was added to 200 µL plasma. After proteins were precipitated with 2 volumes of methanol at –20°C followed by centrifugation at 12,000 x g for 2 minutes, topotecan in a 100 µL aliquot of supernatant was determined by high-performance liquid chromatography. Separation was accomplished on a LiChrosphere RP-100 (125 x 4 mm, 5 µm particle size) cartridge column eluted at a flow rate of 1 mL/min with a mobile phase (apparent pH adjusted to 6.0 with H3PO4) consisting of methanol/water/0.25 mol/L sodium dioctylsulfosuccinate/1.0 mol/L sodium phosphate (pH 6.0)/triethylamine (325:215:20:11.5:1.5). Fluorescence of the column effluent was monitored with excitation and emission wavelengths of 381 and 527 nm, respectively. The assay sensitivity limit and linear range were 1.0 and 1.0 to 20.0 ng/mL, respectively. The interday assay variability was <15% as determined by measuring the coefficient of variation of quality control specimens on 5 separate days over a 2-month period.
To assess carboplatin plasma concentrations, platinum content in the ultrafiltrates was determined in the Mayo Clinic Metals Laboratory by inductively coupled mass spectrometry (37) using known amounts of H2PtCl3 as a standard. From these analyses, plasma Css were calculated as the average of the 24- to 120-hour plasma concentrations. CLss was calculated by dividing the dose rate by Css. CLss values for carboplatin were corrected for the molecular weights of carboplatin (371.25 g/mol) and platinum (195.1 g/mol).
Immunoblotting. Mononuclear cells were isolated from pretreatment bone marrow samples by Ficoll-Hypaque density sedimentation (38). After a wash in RPMI 1640 containing 10 mmol/L HEPES (pH 7.4 at 21°C), aliquots were removed for counting and morphologic analysis. Samples were then solubilized under reducing conditions in lysis buffer containing 6 mol/L guanidine hydrochloride and prepared for electrophoresis as described previously (38). Polypeptides from 5 x 105 cells were applied to adjacent wells of gels containing 5% to 15% (w/v) acrylamide gradient. To provide a standard curve, serial dilutions of HL-60 cells (lyophilized from a large aliquot into multiple single-use vials) were applied to each gel. After electrophoresis, samples were transferred to nitrocellulose and probed with antibodies using techniques recently described in detail (38, 39). These studies employed monoclonal antibodies against topoisomerase I (Y-C. Cheng, Yale University Medical School, New Haven, CT), Chk1 (Santa Cruz Biotechnology, Santa Cruz, CA), checkpoint kinase 2 (Junjie Chen, Mayo Clinic, Rochester, MN), BCRP (George Scheffer, Free University Hospital, Amsterdam, the Netherlands), proliferating cell nuclear antigen (PCNA; Sigma, St. Louis, MO), Bcl-2 (DAKO, Carpinteria, CA), and histone H1 (James Sorace, Veteran's Affairs Medical Center, Baltimore, MD) as well as polyclonal antisera that recognize B23 (40), Mcl-1 (DAKO), Bax (Santa Cruz Biotechnology), and Bak (MBL, Woburn, MA). After blots were scanned, values were corrected for loading differences using the relative content of histone H1 determined in each sample (39). Correlations were analyzed using StatView 5.0 (SAS Institute, Cary, NC). Relationships between relative polypeptide levels and response to therapy were explored graphically using dot plots and evaluated using the Mann-Whitney U test.
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Results |
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1 year in CR, many while still receiving consolidation therapy. Hematologic and infectious toxicities. All patients experienced prolonged grade 4 myelosuppression requiring platelet and RBC support. The WBC typically began to decrease by day 3 of treatment and reached a prolonged nadir of <1,000/mm3 between days 10 and 30. The absolute neutrophil count paralleled the total WBC, typically reached a nadir of <100/mm3 (median, 10; range, 0-380), and, in those who subsequently achieved a CR, remained <500 until days 30 to 35.
Three patients at dose level 1 whose day 14 marrows showed residual leukemia received a second cycle of induction therapy on day 21. Although two of these three patients ultimately achieved a CR, all three had grade 4 neutropenia beyond day 50 after initiation of the second treatment course; and this practice was discontinued.
Consistent with the observed myelosuppression, neutropenic fever occurred in all patients and was treated with empirical antimicrobial regimens. There were six infectious deaths, including three due to respiratory syncitial virus during an epidemic of this agent at one of our institutions, two due to typhlitis (see below), and one due to diverticulitis and peritonitis in a patient who had diverticulitis and fistula formation during previous induction therapy. Two patients at dose level 1 also had nonfatal perirectal infections, one after surgical incision of a thrombosed hemorrhoid while neutropenic and the second after suffering a rectal tear while straining early in her second induction course. After bulk laxatives and stool softeners were added to the supportive care guidelines, there were no further perirectal infections.
The hematologic toxicities of this regimen were substantially less severe during consolidation therapy. Patients receiving consolidation did not become neutropenic until days 12 to 17 of therapy and had briefer periods of grade 4 hematologic toxicity (generally 10-14 days).
Nonhematologic toxicities. Gastrointestinal toxicities were dose limiting. Dose level 4 (1.25 mg/m2/d topotecan and 150 mg/m2/d carboplatin) was well tolerated, with few serious noninfectious toxicities. At dose level 5 (1.3 and 200 mg/m2/d, respectively), one patient experienced fatal typhlitis beginning on day 12 and a second had nonfatal grade 3 stomatitis and grade 4 dysphagia lasting several days. Because this was the first dose level at which carboplatin was increased, the protocol was modified to keep carboplatin constant at 150 mg/m2/d, whereas topotecan was increased further (levels 6 and 7; Table 2). At dose level 7 (2.0 mg/m2/d topotecan and 150 mg/m2/d carboplatin), dose-limiting gastrointestinal toxicity was again observed, with stomatitis in four of five patients (two grade 3 and two grade 4) as well as fatal typhlitis in one patient. Accordingly, dose level 6 (1.6 mg/m2/d topotecan and 150 mg/m2/d carboplatin) was the MTD and recommended phase II dose. At this dose level, one of six patients had grade 4 mucositis during induction. Interestingly, this same patient experienced grade 3 typhlitis while receiving consolidation at dose level 4, suggesting an inability to tolerate one or both agents.
Nonhematologic toxicities also included electrolyte abnormalities (Table 3). These were sporadic, readily reversed with medical intervention, and mild or readily attributable to diuretic or amphotericin administration.
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Pharmacokinetics. Steady-state pharmacokinetics of total topotecan were evaluated in the 29 Mayo Clinic patients enrolled, 24 without prior SCT and 5 with prior SCT. Topotecan CLss was constant over the dose range, with a mean value of 142 ± 43 mL/min/m2. Consequently, topotecan plasma Css increased in proportion to dose over the range of 0.3 to 2.0 mg/m2 (Fig. 1A). Importantly, when individual topotecan measurements in each patient were compared during the 5-day infusion, there was no evidence for an alteration in drug clearance over time (Fig. 1B). This is in contrast to the combination of topotecan plus cisplatin in solid tumor patients, where administration of cisplatin on day 1 diminishes topotecan clearance on subsequent days (41).
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Relationship between pretreatment blast cell protein expression and response. Previous studies have identified several factors that affect sensitivity to topoisomerase I poisons in vitro (reviewed in refs. 26–28, 32). Bone marrow mononuclear cells (median, 80% blasts) isolated from 41 patients before therapy were examined by immunoblotting to assess the potential relationship between some of these factors and clinical response.
To determine whether cells were progressing through the cell cycle, whole-cell lysates were probed for expression of PCNA, a well-characterized marker of cycling cells (42). After normalization for loading using histone H1, a polypeptide present in equal amounts in all diploid cells, PCNA levels varied over a >20-fold range (Fig. 2A and B). PCNA content did not, however, correlate with response (Fig. 2B; P = 0.41, Mann-Whitney U test).
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= 0.83; P < 0.0001) between topoisomerase I and PCNA (Fig. 2C), suggesting that topoisomerase I expression might increase when blasts are actively cycling, as it does when normal lymphohematopoietic cells traverse the cell cycle (43). No correlation between higher topoisomerase I levels and clinical response was observed. To the contrary, there was a weak correlation between low topoisomerase I levels and response (Fig. 2B; P = 0.03, Mann-Whitney U test). Preclinical studies have suggested that the ability to activate a checkpoint pathway sequentially involving the kinases ATR and Chk1 can affect sensitivity to topoisomerase I poisons (reviewed in refs. 28, 32) as well as platinating agents (44). In our samples, ATR expression was barely detectable and was not quantifiable (Fig. 2A). Chk1 was readily detectable in most samples (Fig. 2A) but failed to correlate with response. Instead, Chk1 levels correlated with PCNA (Fig. 2C), consistent with previous results showing cell cycle–dependent Chk1 expression (45).
The transporter BCRP has been implicated in topotecan resistance in tissue culture (26, 28, 30). This polypeptide was detectable in 38% of samples analyzed (Fig. 2A) but also did not correlate with response.
Finally, Bcl-2 family members have been reported to affect sensitivity to topoisomerase I poisons as well as cisplatin in preclinical models (34). Surprisingly, levels of Mcl-1, Bax, and Bak correlated with PCNA (Table 5) rather than response. In contrast, there was a correlation between low Bcl-2 content and response to therapy (Fig. 2A and B; P = 0.014, Mann-Whitney U test).
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Discussion |
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When carboplatin and topotecan were administered by 5-day continuous infusion, the MTD in adults with relapsed/refractory leukemia was 150 mg/m2/d carboplatin plus 1.6 mg/m2/d topotecan. Prolonged neutropenia extending to days 30 to 35 was observed in all patients who achieved a PR or CR. Gastrointestinal toxicity, including typhlitis as well as severe oral and perianal mucositis, was dose limiting. Thus, the current regimen must be administered with caution, although its toxicities are similar to those observed with other salvage regimens, such as mitoxantrone, etoposide, and cytarabine (47).
Previous studies of topotecan-containing regimens in patients with refractory and relapsed acute leukemia have been somewhat disappointing. As a single agent, topotecan induced CRs in only 4 of 44 adults when administered as a 5-day continuous infusion in phase I studies to populations similar to those enrolled in the present trial (12, 13). Addition of cytarabine or etoposide to topotecan failed to substantially improve the CR rate (11). Because preclinical studies suggested that DNA-damaging agents might better augment topotecan cytotoxicity (23), the present study examined the effect of combining carboplatin with topotecan. The overall CR rate of 15% (7 of 46 CR) with this regimen is similar to the CR rate of 17% observed in a phase I trial of a regimen containing cyclophosphamide plus cytarabine plus topotecan (11). This overall response rate is also similar to that of single-agent carboplatin (8–10). It is important to note, however, that most responses in the present trial occurred at the highest two dose levels, including three CRs and one PR in six patients treated at the MTD. A phase II study would be required to determine whether the response rate observed at the MTD is maintained in a larger cohort.
Laboratory studies done in conjunction with the present trial focused on two main issues. First, in view of data suggesting that cisplatin affects topotecan clearance in solid tumor patients (41), the effect of carboplatin on steady-state topotecan levels was assessed. Because topotecan levels were constant throughout the duration of the 5-day infusion (Fig. 1B) and were similar to levels observed during administration of single-agent topotecan (13), we conclude that carboplatin did not diminish topotecan clearance.
Second, in view of increasing information about potential mechanisms of resistance to topotecan (26–28), we examined expression of a variety of polypeptides in blasts harvested before treatment. Because this study was almost complete before it was reported that drug-induced topoisomerase I down-regulation correlates with drug sensitivity in preclinical models (28, 33), the present trial did not include procurement of bone marrow specimens after treatment was initiated. Instead, the present analysis focused on pretreatment leukemic cell characteristics that might predict response.
These studies showed that BCRP, which is capable of effluxing topotecan and SN-38 (28, 30), is detectable by immunoblotting in 
40% of relapsed and refractory leukemias (Fig. 2A). Interestingly, however, BCRP expression did not correlate with response. This might reflect the inclusion of carboplatin, an agent that is unaffected by BCRP, or the possible ability of immunologic methods to detect BCRP at levels below those that significantly affect topotecan accumulation (31).
Recent studies have suggested that platinating agents (44) and topoisomerase I poisons (reviewed in refs. 28, 32) activate a DNA damage–induced signal transduction pathway involving Chk1 and downstream targets. There was, however, no correlation between Chk1 expression and response. Instead, consistent with earlier experiments in tissue culture cells (45), Chk1 levels correlated with expression of PCNA, a marker of proliferation. Because the proliferative state of the bulk of the leukemia cells present in the pretreatment marrow might not be representative of the leukemia stem cells, it is not surprising that the levels of PCNA and Chk1 failed to correlate with response.
When levels of Bcl-2 family members were examined, expression of Mcl-1, Bax, and Bak also correlated with PCNA (Table 5). To our knowledge, this is the first suggestion that Mcl-1 levels correlate with proliferation, although previous studies have found that Mcl-1 message and polypeptide levels are diminished in cells treated with inhibitors of the mitogen-activated protein kinase pathway (48, 49), a pathway that drives cell proliferation.
In contrast, Bcl-2 levels did not correlate with PCNA (Table 5). Instead, patients with leukemia expressing low amounts of Bcl-2 were more likely to respond to the topotecan/carboplatin regimen (Fig. 2B). This observation complements and extends previous experiments showing that Bcl-2 overexpression confers resistance to camptothecin and cisplatin in tissue culture cells (26, 27, 34). Because of the small number of patients responding in the present trial, further studies in a larger patient cohort are required to confirm the association between low Bcl-2 expression and response. Nonetheless, these results suggest that the response to the topotecan/carboplatin regimen might be enhanced by addition of agents such as HA14-1 (50), which inhibit Bcl-2 function. Additional preclinical studies are required to assess this possibility.
In summary, results of the present study identify gastrointestinal toxicity as dose limiting when leukemia patients without prior SCT receive topotecan plus carboplatin induction therapy, identify the MTD as 1.6 mg/m2/d topotecan and 150 mg/m2/d carboplatin when these agents are administered by 5-day continuous infusion, show that responses are more frequent at doses close to the MTD, and suggest that diminished expression of Bcl-2 is a potential predictive factor with this regimen.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: J.E. Karp and J. Greer are currently at the Sidney Kimmel Cancer Center at Johns Hopkins, Baltimore, MD.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4/12/05; revised 6/ 1/05; accepted 6/ 6/05.
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