Phase II Clinical Trial of Ixabepilone (BMS-247550), an Epothilone B Analog, in Metastatic and Locally Advanced Breast Cancer
http://www.100md.com
《临床肿瘤学》
the Cancer Therapeutics Branch, Medical Oncology Clinical Research Unit, and Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD
the Magee-Women's Hospital, University of Pittsburgh Cancer Institute, Pittsburgh, PA
ABSTRACT
PURPOSE: Ixabepilone (BMS-247550) is an epothilone B analog that stabilizes microtubules and has antitumor activity in taxane-refractory patients in phase I studies. In a phase II trial, we evaluated the efficacy and safety of ixabepilone in women with metastatic and locally advanced breast cancer.
PATIENTS AND METHODS: Breast cancer patients with measurable disease who had paclitaxel and/or docetaxel as prior neoadjuvant, adjuvant, or metastatic therapy were treated with ixabepilone at 6 mg/m2/d intravenously on days 1 through 5 every 3 weeks. Levels of glutamate (glu) -terminated and acetylated -tubulin, markers of microtubule stabilization, were detected by Western blot and by immunohistochemistry in a subset of matched pre- and post-treatment tumor biopsies.
RESULTS: Thirty-seven patients received 153 cycles of ixabepilone. The best responses were a complete response in one patient (3%), partial responses in seven patients (19%), and stable disease in 13 patients (35%). Grade 3 and 4 toxicities included neutropenia (35%), febrile neutropenia (14%), fatigue (14%), diarrhea (11%), nausea/vomiting (5%), myalgia/arthralgia (3%), and sensory neuropathy (3%). Two patients were removed from study because of prolonged grade 2 or 3 neurotoxicity, and three patients were removed from study for other grade 3 and 4 nonhematologic toxicities. Compared with baseline levels, levels of both glu-terminated and acetylated -tubulin were increased in tumor biopsies performed after ixabepilone therapy.
CONCLUSION: An objective response was seen in 22% of the patients in a population who had been previously treated with a taxane. Sensory neuropathy was mild with grade 3 neurotoxicity rarely seen. Microtubule stabilization occurred in tumor biopsies after treatment with ixabepilone.
INTRODUCTION
The microtubule-stabilizing drugs paclitaxel and docetaxel are considered among the most effective chemotherapeutic agents for the treatment of metastatic breast cancer. 1 However, during the course of treatment of metastatic disease, even tumors that initially respond to these drugs eventually develop resistance, and the sequential use of other chemotherapeutics is standard practice. For many patients receiving paclitaxel and docetaxel, sensory peripheral neuropathy can be significant and even dose limiting. New therapies with microtubule-stabilizing activity are being investigated for their potential activity in the treatment of cancer, 2 and have generated considerable interest for the treatment of metastatic breast cancer.
Microtubules are filaments consisting of - and -tubulin. Microtubule homeostasis is characterized by dynamic polymerization and depolymerization of the tubulin subunits. Disruption of microtubule function through increased stability can lead to cell cycle arrest and apoptosis. Microtubule stabilization results in a number of post-translational changes to -tubulin, 3 including the removal of the carboxy-terminal tyrosine to reveal a terminal glutamate (glu-terminated -tubulin), 4 and acetylation of the lysine 40 (acetylated -tubulin). 5 Detyrosination, a potential marker for tumor aggressiveness, 6 occurs only on polymerized microtubules, whereas its restoration occurs exclusively on soluble -tubulin heterodimers. Tubulin acetylation occurs only after polymerization; depolymerized tubulin is rapidly deacetylated in vivo. 7 Microtubule-stabilizing agents reduce microtubule dynamics and promote tubulin polymerization. 8
The epothilones are macrolide fermentation products of the myxobacterium Sorangium cellulosum. Epothilones A and B competitively displace paclitaxel from microtubules in vitro, and similar to paclitaxel, stabilize microtubules and cause cell cycle arrest and cytotoxicity. 9 Ixabepilone (BMS-247550) is a semisynthetic analog of epothilone B, in which the lactone oxygen of epothilone B is replaced by nitrogen, increasing the stability of the drug. 10 In preclinical models, ixabepilone was active in the paclitaxel-resistant breast cancer cell line Pat-21 and in other paclitaxel-insensitive models. 11
A phase I study established that ixabepilone administered at 6 mg/m2/d on 5 consecutive days every 3 weeks was the maximum-tolerated dose; neutropenia was the dose-limiting toxicity. 12 This regimen was considered particularly attractive because the longer cycle administration time had less neurotoxicity than administration schedules used in other phase I trials of ixabepilone.
We conducted a phase II trial of ixabepilone for patients with metastatic and locally advanced breast cancer, with the primary objectives of determining the efficacy and safety of ixabepilone. Our secondary objectives included assessing ixabepilone activity at the tumor site by evaluating microtubule stabilization within the target tissue. Using tumor biopsies collected at baseline and during treatment (day 2 of cycle 2), we quantified -tubulin acetylation and glu-termination to determine whether baseline levels predicted response to ixabepilone and whether changes during treatment correlated with response.
PATIENTS AND METHODS
Eligibility
Eligible patients had a diagnosis of metastatic or locally advanced breast adenocarcinoma confirmed by the pathology department of the enrolling institution; an Eastern Cooperative Oncology Group performance status of 0, 1, or 2; and measurable disease by Response Criteria in Solid Tumors (RECIST) criteria. 13 Laboratory values were required to be within the specified ranges within 1 week of study enrollment, including an absolute neutrophil count of 1.5 x 109/L and thrombocyte count of 100 x 109/L. All patients had received at least one prior neoadjuvant, adjuvant, or metastatic regimen that contained docetaxel or paclitaxel; otherwise, there were no other restrictions on prior number of therapies, including anthracycline-containing or high-dose chemotherapy regimens. Patients with nonmetastatic locally advanced breast cancer had received and not responded to prior anthracycline and taxane neoadjuvant chemotherapy. Because of concerns that ixabepilone metabolism may be inhibited by potent cytochrome P450 3A4 inhibitors, patients were also excluded from receiving the following medications at enrollment and while enrolled onto the study: amiodarone, clarithromycin, erythromycin, fluconazole, itraconazole, ketoconazole, indinavir, nelfinavir, ritonavir, and saquinavir. Patients with grade 2 or greater neuropathy at baseline were excluded.
Study Design and Treatment Modifications
This phase II study was approved by the institutional review board (IRB) for the Center for Cancer Research, National Cancer Institute (NCI), and by the IRB of the Magee-Women's Hospital (Pittsburgh, PA). All patients signed the IRB-approved informed consent before participating.
Ixabepilone (provided by the Cancer Therapy Evaluation Program, NCI) was administered during 1 hour intravenously (IV) on 5 consecutive days every 3 weeks. All patients received a starting dose of 6 mg/m2/d for the first cycle. All patients received premedication with diphenhydramine at 50 mg IV or orally, or hydroxyzine at 25 mg orally, and ranitidine at 50 mg IV, 30 to 60 minutes before each dose of ixabepilone. Prophylactic antiemetics were not routinely administered, but were added to the regimen for patients experiencing toxicity. An absolute neutrophil count 1.0 x 109/L and a thrombocyte count 75 x 109/L was required on the first day of treatment of each cycle.
Toxicities were assessed using NCI Common Toxicity Criteria version 2.0 14 at baseline and at the conclusion of each cycle of treatment. Dose reductions to 5 mg/m2/d and then to 4 mg/m2/d were implemented for patients experiencing grade 3 and 4 nonhematologic toxicities during the prior cycle, febrile neutropenia, or for toxicities requiring a delay in treatment. Toxicities (including neurotoxicity) required resolution to grade 1 or to baseline before the next cycle of treatment was administered. Granulocyte colony-stimulating factors were not given initially at cycle 1, but could be added to subsequent cycles of treatment for patients with febrile neutropenia or delayed neutrophil recovery requiring dose delay. Treatment intervals could be extended up to 5 weeks between the first days of treatment for patients with optimal responses who had completed six cycles of treatment. Patients were removed from study for the following reasons: disease progression, persistent grade 2 neuropathy or grade 3 neuropathy lasting more than 7 days, grade 3 or 4 toxicities requiring more than two dose reductions or delay of treatment for more than 5 weeks from the beginning of the last cycle, or per patient request.
Response Assessment
Measurable disease was assessed by imaging using the RECIST criteria. 13 Patients underwent baseline imaging within 4 weeks of enrollment, and were scanned before every other cycle. Stable disease was determined before cycle 3 at 6 weeks. All partial and complete responses were confirmed at least 4 weeks later with repeat imaging.
Statistical Design and Methods
This protocol was designed to evaluate the efficacy of ixabepilone. The study used an optimal two-stage design to rule out a low 5% response rate in favor of a 20% response rate. 15 With a 10% probability of accepting a poor agent and rejecting a good agent, if at least one of the first 12 patients had a response, enrollment would continue until 37 patients were enrolled. Four or more responses were considered consistent with an active agent worthy of additional development. Under the null hypothesis (5% response probability), the study had a 60% probability of early termination.
Time to progression was calculated for all patients enrolled from the first day of treatment to the day of evidence of progressive disease, with censoring at last follow-up (without progression), or removal from study for toxicity. The one ineligible patient was censored at the time of enrollment. Progression-free survival probabilities as a function of time were calculated using the Kaplan-Meier method. Duration of response was calculated for the eight patients who had confirmed responses, and was calculated from the day of first radiologic partial response until the date of progression. The one remaining patient enrolled onto the study was censored at the date of her last visit, August 25, 2004.
Correlative Studies
Patients with tumors that could be biopsied under local anesthesia had core or punch biopsies obtained at baseline (before receiving the first dose of ixabepilone), and again on cycle 2 day 2, approximately 18 to 24 hours after the first dose of cycle 2. The biopsy timing was chosen to allow enough time (after a full cycle, but less than 24 hours after a treatment infusion) for ixabepilone to theoretically have a discernible target effect.
For Western blot analysis, frozen tissues were thawed, macerated, and dounce-homogenized in less than 1 mL of a hypotonic lysis buffer containing 1 mmol/L MgCl2, 2 mmol/L ethyleneglycoltetraacetic acid, 0.5% Nonidet P-40 (Amersham Biosciences, Piscataway, NJ), 20 mmol/L Tris-HCl, pH 6.8, aprotinin (200 units/mL), and an additional EDTA-free protease inhibitor-mix tablet (Roche Diagnostics, Mannheim, Germany). Tissue lysates were vortexed and centrifuged to remove unlysed cells and tissue debris. Equal amounts of protein (10 to 25 μg) were run on 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to Immobilon (Millipore, Billerica, MA) membranes. The membranes were blocked, and sequentially probed with a mouse monoclonal antibody to -actin (Sigma Chemical Co, St Louis, MO) as a loading control, mouse monoclonal anti--tubulin clone DM1A (Sigma), mouse monoclonal antiacetylated -tubulin, clone 6-11B-1 (Sigma), and rabbit antidetyrosinated tubulin polyclonal antibody (Chemicon International Inc, Temecula, CA). The membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies, with appropriate species reactivity, and the protein-antibody complexes were detected by using Super-Signal Chemiluminescent reagents (Pierce Biochemicals, Rockford, IL) and quantitated by densitometry.
For immunohistochemistry, formalin-fixed, paraffin-embedded sections were processed as described previously. 16 Acetylated and glu-terminated mouse monoclonal -tubulin antibodies (described above), and mouse monoclonal anti-Ki67 (MIB-1) antibody (Dako, Carpinteria, CA) were applied to tissue sections at a dilution of 1:1000, 1:1000, and 1:50, respectively. The sections were scored quantitatively using the Automated Cellular Imaging System (ACIS; Chromavision, San Juan Capistrano, CA). Six areas of each tumor were scored using a free-scoring or 40x magnification tool to generate an averaged percentage and intensity of stained tumor cells. The staining index was calculated by multiplying the percentage of positively stained cells by the average staining intensity after subtracting the machine readouts of the corresponding negative control for each marker. For Ki67, a labeling percentage was reported.
RESULTS
Patients
In this phase II study, 37 patients were enrolled at the NCI (29 patients) or at the University of Pittsburgh Cancer Institute (eight patients) between June 2002 and March 2004. All patients received at least one cycle and all patients are included in the analysis.
The baseline characteristics of the patients are summarized in Table 1. All patients had at least two cycles of a paclitaxel- or docetaxel-containing regimen (as neoadjuvant, adjuvant, or metastatic therapy) at some point in their therapeutic history. Twenty-two (59%) patients had progressive disease while receiving paclitaxel- and/or docetaxel-containing regimens.
Patients received a median of four cycles (range, one to 11 cycles) of ixabepilone chemotherapy. Of the 37 patients enrolled, 29 patients were removed from study for disease progression, two patients were removed after maximum benefit was achieved (one for neoadjuvant therapy and one for resolution of nonmeasurable disease), five patients were removed for prolonged toxicity, and one patient remains enrolled onto the study.
Efficacy
The objective response was 22% (95% CI, 9.8% to 38.2%), with one complete response and seven partial responses. Stable disease for at least 6 weeks was the best response for 35% of patients (13 patients). The median time to progression for all patients was 80 days (Fig 1). Patients classified as having progressive disease included one patient enrolled and treated with good clinical response but determined to be ineligible retrospectively when found to be without measurable disease by RECIST criteria, one patient with severe toxicity during the first cycle who refused additional evaluation, and two patients who were considered to have progressive disease 8 and 10 days after starting therapy because of life-threatening tumor complications.
For eight patients with confirmed partial responses, the median response duration was 118 days. One patient with a partial response at 6 weeks improved to a confirmed complete response after 8 months (250 days). Measurable responses were seen in lung and liver lesions, and supraclavicular, mediastinal, and axillary lymph nodes. Improvements were also seen in nontarget lesions, such as breast and bone lesions and pleural effusions. Responses were seen in patients who had experienced disease progression after receiving a prior taxane and well as patients previously exposed to an anthracycline, capecitabine, and a taxane (Table 2). One of two patients enrolled who had experienced disease progression after receiving both paclitaxel and docetaxel had a partial response to ixabepilone. One patient with inflammatory breast cancer who had stable disease with neoadjuvant docetaxel and doxorubicin was switched to, and responded to, ixabepilone as a neoadjuvant therapy.
Toxicities
Toxicity information was collected for all patients, who received a total of 153 cycles of therapy. For each patient, the worst grades seen of the most common hematologic and nonhematologic toxicities are summarized in Table 3. One patient was removed from study because of grade 3 sensory neuropathy and one patient was removed from study because of prolonged grade 2 sensory neuropathy. During their first cycle of ixabepilone, two patients had multiple grade 3 and 4 toxicities, including febrile neutropenia, thrombocytopenia, severe mucositis, diarrhea, fatigue, and sensory neuropathy. Neither of these patients received additional treatment with ixabepilone; one of these two patients received concurrent warfarin and clarithromycin, whereas the other did not.
One patient developed slowly progressive patchy pulmonary infiltrates that showed an organizing, nonspecific interstitial pneumonia and metastatic tumor cells on lung biopsy. She was removed from the study, and after receiving high-dose corticosteroids the infiltrate resolved symptomatically and radiologically.
Twelve patients required dose reduction while enrolled onto the study (median, cycle 5; range, cycles 2 to 10). The dose was reduced because of neuropathy in six patients, and because of diarrhea, fatigue, and neutropenia in two patients each. Only one patient required a second dose reduction because of a grade 3 myalgia.
Five patients received erythropoietin or darbepoetin. Four patients received filgrastim or pegfilgrastim for febrile neutropenia. Three patients received prophylactic pegfilgrastim because of prolonged neutrophil recovery in a prior cycle or for a prior episode of febrile neutropenia.
Peripheral Sensory Neuropathy
Of the 24 patients with no baseline neuropathy, 14 patients (58%) developed grade 1, six patients (25%) developed grade 2, and one patient (4%) developed grade 3 peripheral sensory neuropathy as the worst grade of neuropathy. Only two (15%) of 13 patients with baseline grade 1 neuropathy developed grade 2 neuropathy. In total, nine patients developed at least grade 2 neuropathy during the course of their treatment; two of these patients developed grade 2 neuropathy during the first cycle, along with other severe hematologic and nonhematologic toxicities. Of the other seven patients, the median time to development of grade 2 neuropathy was 108 days on study (range, 42 to 189 days). The median times to development and resolution of neuropathy and the outcomes are summarized in Figure 2.
Correlative Studies
Biopsies were collected at baseline and during cycle 2 from five patients (one patient had partial response, two patients had stable disease, and two patients had progressive disease) and were confirmed by pathology to contain tumor. Four of these paired patient biopsies were analyzed by Western blot. For one patient (patient 5), the baseline sample was not adequate. Increased acetylation of -tubulin was seen by Western blot for all four patients (labeled patients 1 through 4 with their responses in Fig 3). Glu-termination was also increased in patients 2, 3, and 4.
Because the protein lysates run on Western blot contain both tumor and stromal components of the biopsy, we next chose to quantify tumor changes alone by immunohistochemistry. Four of the five paired patient biopsies (labeled patients 1, 2, 3, and 5) had tissue available for sectioning for immunohistochemistry. For patient 4, no tumor remained from the baseline paraffin block. For patient 1, who had a partial response to ixabepilone, levels of acetylated -tubulin were detected in both tumor and stromal cells in the baseline sample and increased in both cellular compartments with treatment (Figs 4A and 4B). In contrast, for patient 5, who had progressive disease, levels of acetylated -tubulin were detected only in stromal cells at baseline and increased in the stroma but were still absent in the tumor cells with treatment (Figs 4C and 4D). Baseline levels of acetylated -tubulin were present before treatment and increased after treatment in the patients with response and stable disease (Fig 5A), and levels of glu-terminated -tubulin increased in all patients with treatment (Fig 5B). Ki67 labeling percentage, a measure of tumor proliferation, decreased in the same two patients with increased levels of acetylated -tubulin after treatment, but the Ki67 labeling percentage remained similar in the two patients with no change in the level of acetylated -tubulin after treatment (Fig 5C).
DISCUSSION
Ixabepilone has been studied in a number of phase I and II clinical trials, including one phase I and three phase II breast cancer trials (not including the current study) that have been presented previously in abstract form. Partial response rates for the phase II clinical trials have been 12% for a taxane-resistant population, 17 44% for a population with prior adjuvant or neoadjuvant anthracycline therapy and no prior therapy for metastatic disease, 18 and 31% for another mixed population of breast cancer patients. 19 Because our patients received taxanes in a variety of prior regimens, including neoadjuvant or adjuvant therapy, and the number of prior treatments for metastatic disease varied widely, it is not surprising that our response rate of 22% falls within the range of response rates previously reported. In metastatic breast cancer patients with docetaxel-resistant disease, weekly paclitaxel had a 32% response rate, 20 whereas in patients with paclitaxel-resistant disease, docetaxel had an 18% response rate. 21
In our study, there was a low incidence of hematologic toxicities seen with this schedule, and the most significant complaints were fatigue, myalgia, and peripheral sensory neuropathy. However, for two patients, significant toxicities occurred in the first cycle requiring cessation of additional therapy. In both patients severe febrile neutropenia, nausea, vomiting, diarrhea, mucositis, and peripheral neuropathies occurred within a week of completing the first cycle of treatment. The reason for the comparatively severe toxicities seen in these two patients is unknown. One patient, who had been extensively treated with docetaxel previously without significant complications, received clarithromycin (a cytochrome P450 3A4 inhibitor) on day 4 of cycle 1, leading us to speculate whether a drug-drug interaction may have been responsible for symptoms. However, the second patient did not receive any cytochrome P450 inhibitors. Both patients received warfarin, as did three other patients without severe toxicities. Because blood sampling was not done with this study, pharmacokinetic information regarding drug levels was not available.
One of the most notable complications associated with taxane and other microtubule-targeted treatments is the development of sensory peripheral neuropathy. Although most of our patients who received four or more cycles of ixabepilone had some subjective symptoms of neuropathy, these usually did not interfere with function. Only one patient was considered to have grade 3 neurotoxicity because her peripheral neuropathy was the primary reason she felt compelled to stop working. Grade 3 neurotoxicity was not seen in the phase I study with the regimen administered daily for 5 days. 12 The grade 3 peripheral sensory neuropathy seen with ixabepilone at this schedule (3%) appears to be less than that reported for other ixabepilone schedules (4% to 25%). 19, 22- 26 The lower incidence of neuropathy may be related to the administration schedule, or to the population enrolled onto this study. In our study, all of our patients had received a taxane previously, which may make them more susceptible to either developing a neuropathy or to having a recurrence of previous neurotoxicity. However, because all patients were either considered grade 0 or 1 at study entry, patients who already had a significant unresolved neuropathy would not have been enrolled. For similar reasons, a patient with a history of significant peripheral neuropathy may have been less willing to enroll onto this trial.
the available paired tumor biopsies, we observed increased levels of both glu-terminated and acetylated -tubulin after treatment, indicating that ixabepilone stabilized microtubules in the target tissue. Measures of stabilized microtubules may not only demonstrate a drug effect, but may also help to predict response when examined in a pretreatment specimen. Interestingly, we found that acetylated -tubulin levels were higher at baseline in the tumor cells of patients whose tumors responded or in patients with stable disease than in the patient whose tumor did not respond. In the patient with progressive disease, baseline levels of acetylated -tubulin within the tumor were not detectable by immunohistochemistry. Thus, our results suggest that a tumor with inherent microtubule stability might be more likely to respond to a stabilizing agent with additional microtubule stabilization and decreased proliferation. Although the small numbers of patients biopsied in this group limits our ability to make more definitive statements, these interesting observations are hypothesis generating and should be considered for additional study.
In summary, ixabepilone has shown activity in patients with metastatic breast cancer who have been previously treated with paclitaxel or docetaxel. For most patients, ixabepilone was well tolerated with low hematologic toxicity, and minimal or easily controlled nausea, vomiting, and diarrhea. In addition, administration of ixabepilone does not require premedication with corticosteroids, unlike paclitaxel and docetaxel, which improves its tolerability. Neurotoxicity, a major concern for microtubule-stabilizing drugs, was relatively mild for ixabepilone on this schedule. Its activity and safety profile warrant additional development of this therapy.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Adam Brufsky, Bristol-Myers Squibb. Honoraria: Adam Brufsky, Bristol-Myers Squibb. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Diana Nguyen for immunohistochemistry and A. Dimitrios Colevas, MD, for helpful discussions regarding the conduct of this trial.
NOTES
Presented at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004 (preliminary portions of the study, abstract 545).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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the Magee-Women's Hospital, University of Pittsburgh Cancer Institute, Pittsburgh, PA
ABSTRACT
PURPOSE: Ixabepilone (BMS-247550) is an epothilone B analog that stabilizes microtubules and has antitumor activity in taxane-refractory patients in phase I studies. In a phase II trial, we evaluated the efficacy and safety of ixabepilone in women with metastatic and locally advanced breast cancer.
PATIENTS AND METHODS: Breast cancer patients with measurable disease who had paclitaxel and/or docetaxel as prior neoadjuvant, adjuvant, or metastatic therapy were treated with ixabepilone at 6 mg/m2/d intravenously on days 1 through 5 every 3 weeks. Levels of glutamate (glu) -terminated and acetylated -tubulin, markers of microtubule stabilization, were detected by Western blot and by immunohistochemistry in a subset of matched pre- and post-treatment tumor biopsies.
RESULTS: Thirty-seven patients received 153 cycles of ixabepilone. The best responses were a complete response in one patient (3%), partial responses in seven patients (19%), and stable disease in 13 patients (35%). Grade 3 and 4 toxicities included neutropenia (35%), febrile neutropenia (14%), fatigue (14%), diarrhea (11%), nausea/vomiting (5%), myalgia/arthralgia (3%), and sensory neuropathy (3%). Two patients were removed from study because of prolonged grade 2 or 3 neurotoxicity, and three patients were removed from study for other grade 3 and 4 nonhematologic toxicities. Compared with baseline levels, levels of both glu-terminated and acetylated -tubulin were increased in tumor biopsies performed after ixabepilone therapy.
CONCLUSION: An objective response was seen in 22% of the patients in a population who had been previously treated with a taxane. Sensory neuropathy was mild with grade 3 neurotoxicity rarely seen. Microtubule stabilization occurred in tumor biopsies after treatment with ixabepilone.
INTRODUCTION
The microtubule-stabilizing drugs paclitaxel and docetaxel are considered among the most effective chemotherapeutic agents for the treatment of metastatic breast cancer. 1 However, during the course of treatment of metastatic disease, even tumors that initially respond to these drugs eventually develop resistance, and the sequential use of other chemotherapeutics is standard practice. For many patients receiving paclitaxel and docetaxel, sensory peripheral neuropathy can be significant and even dose limiting. New therapies with microtubule-stabilizing activity are being investigated for their potential activity in the treatment of cancer, 2 and have generated considerable interest for the treatment of metastatic breast cancer.
Microtubules are filaments consisting of - and -tubulin. Microtubule homeostasis is characterized by dynamic polymerization and depolymerization of the tubulin subunits. Disruption of microtubule function through increased stability can lead to cell cycle arrest and apoptosis. Microtubule stabilization results in a number of post-translational changes to -tubulin, 3 including the removal of the carboxy-terminal tyrosine to reveal a terminal glutamate (glu-terminated -tubulin), 4 and acetylation of the lysine 40 (acetylated -tubulin). 5 Detyrosination, a potential marker for tumor aggressiveness, 6 occurs only on polymerized microtubules, whereas its restoration occurs exclusively on soluble -tubulin heterodimers. Tubulin acetylation occurs only after polymerization; depolymerized tubulin is rapidly deacetylated in vivo. 7 Microtubule-stabilizing agents reduce microtubule dynamics and promote tubulin polymerization. 8
The epothilones are macrolide fermentation products of the myxobacterium Sorangium cellulosum. Epothilones A and B competitively displace paclitaxel from microtubules in vitro, and similar to paclitaxel, stabilize microtubules and cause cell cycle arrest and cytotoxicity. 9 Ixabepilone (BMS-247550) is a semisynthetic analog of epothilone B, in which the lactone oxygen of epothilone B is replaced by nitrogen, increasing the stability of the drug. 10 In preclinical models, ixabepilone was active in the paclitaxel-resistant breast cancer cell line Pat-21 and in other paclitaxel-insensitive models. 11
A phase I study established that ixabepilone administered at 6 mg/m2/d on 5 consecutive days every 3 weeks was the maximum-tolerated dose; neutropenia was the dose-limiting toxicity. 12 This regimen was considered particularly attractive because the longer cycle administration time had less neurotoxicity than administration schedules used in other phase I trials of ixabepilone.
We conducted a phase II trial of ixabepilone for patients with metastatic and locally advanced breast cancer, with the primary objectives of determining the efficacy and safety of ixabepilone. Our secondary objectives included assessing ixabepilone activity at the tumor site by evaluating microtubule stabilization within the target tissue. Using tumor biopsies collected at baseline and during treatment (day 2 of cycle 2), we quantified -tubulin acetylation and glu-termination to determine whether baseline levels predicted response to ixabepilone and whether changes during treatment correlated with response.
PATIENTS AND METHODS
Eligibility
Eligible patients had a diagnosis of metastatic or locally advanced breast adenocarcinoma confirmed by the pathology department of the enrolling institution; an Eastern Cooperative Oncology Group performance status of 0, 1, or 2; and measurable disease by Response Criteria in Solid Tumors (RECIST) criteria. 13 Laboratory values were required to be within the specified ranges within 1 week of study enrollment, including an absolute neutrophil count of 1.5 x 109/L and thrombocyte count of 100 x 109/L. All patients had received at least one prior neoadjuvant, adjuvant, or metastatic regimen that contained docetaxel or paclitaxel; otherwise, there were no other restrictions on prior number of therapies, including anthracycline-containing or high-dose chemotherapy regimens. Patients with nonmetastatic locally advanced breast cancer had received and not responded to prior anthracycline and taxane neoadjuvant chemotherapy. Because of concerns that ixabepilone metabolism may be inhibited by potent cytochrome P450 3A4 inhibitors, patients were also excluded from receiving the following medications at enrollment and while enrolled onto the study: amiodarone, clarithromycin, erythromycin, fluconazole, itraconazole, ketoconazole, indinavir, nelfinavir, ritonavir, and saquinavir. Patients with grade 2 or greater neuropathy at baseline were excluded.
Study Design and Treatment Modifications
This phase II study was approved by the institutional review board (IRB) for the Center for Cancer Research, National Cancer Institute (NCI), and by the IRB of the Magee-Women's Hospital (Pittsburgh, PA). All patients signed the IRB-approved informed consent before participating.
Ixabepilone (provided by the Cancer Therapy Evaluation Program, NCI) was administered during 1 hour intravenously (IV) on 5 consecutive days every 3 weeks. All patients received a starting dose of 6 mg/m2/d for the first cycle. All patients received premedication with diphenhydramine at 50 mg IV or orally, or hydroxyzine at 25 mg orally, and ranitidine at 50 mg IV, 30 to 60 minutes before each dose of ixabepilone. Prophylactic antiemetics were not routinely administered, but were added to the regimen for patients experiencing toxicity. An absolute neutrophil count 1.0 x 109/L and a thrombocyte count 75 x 109/L was required on the first day of treatment of each cycle.
Toxicities were assessed using NCI Common Toxicity Criteria version 2.0 14 at baseline and at the conclusion of each cycle of treatment. Dose reductions to 5 mg/m2/d and then to 4 mg/m2/d were implemented for patients experiencing grade 3 and 4 nonhematologic toxicities during the prior cycle, febrile neutropenia, or for toxicities requiring a delay in treatment. Toxicities (including neurotoxicity) required resolution to grade 1 or to baseline before the next cycle of treatment was administered. Granulocyte colony-stimulating factors were not given initially at cycle 1, but could be added to subsequent cycles of treatment for patients with febrile neutropenia or delayed neutrophil recovery requiring dose delay. Treatment intervals could be extended up to 5 weeks between the first days of treatment for patients with optimal responses who had completed six cycles of treatment. Patients were removed from study for the following reasons: disease progression, persistent grade 2 neuropathy or grade 3 neuropathy lasting more than 7 days, grade 3 or 4 toxicities requiring more than two dose reductions or delay of treatment for more than 5 weeks from the beginning of the last cycle, or per patient request.
Response Assessment
Measurable disease was assessed by imaging using the RECIST criteria. 13 Patients underwent baseline imaging within 4 weeks of enrollment, and were scanned before every other cycle. Stable disease was determined before cycle 3 at 6 weeks. All partial and complete responses were confirmed at least 4 weeks later with repeat imaging.
Statistical Design and Methods
This protocol was designed to evaluate the efficacy of ixabepilone. The study used an optimal two-stage design to rule out a low 5% response rate in favor of a 20% response rate. 15 With a 10% probability of accepting a poor agent and rejecting a good agent, if at least one of the first 12 patients had a response, enrollment would continue until 37 patients were enrolled. Four or more responses were considered consistent with an active agent worthy of additional development. Under the null hypothesis (5% response probability), the study had a 60% probability of early termination.
Time to progression was calculated for all patients enrolled from the first day of treatment to the day of evidence of progressive disease, with censoring at last follow-up (without progression), or removal from study for toxicity. The one ineligible patient was censored at the time of enrollment. Progression-free survival probabilities as a function of time were calculated using the Kaplan-Meier method. Duration of response was calculated for the eight patients who had confirmed responses, and was calculated from the day of first radiologic partial response until the date of progression. The one remaining patient enrolled onto the study was censored at the date of her last visit, August 25, 2004.
Correlative Studies
Patients with tumors that could be biopsied under local anesthesia had core or punch biopsies obtained at baseline (before receiving the first dose of ixabepilone), and again on cycle 2 day 2, approximately 18 to 24 hours after the first dose of cycle 2. The biopsy timing was chosen to allow enough time (after a full cycle, but less than 24 hours after a treatment infusion) for ixabepilone to theoretically have a discernible target effect.
For Western blot analysis, frozen tissues were thawed, macerated, and dounce-homogenized in less than 1 mL of a hypotonic lysis buffer containing 1 mmol/L MgCl2, 2 mmol/L ethyleneglycoltetraacetic acid, 0.5% Nonidet P-40 (Amersham Biosciences, Piscataway, NJ), 20 mmol/L Tris-HCl, pH 6.8, aprotinin (200 units/mL), and an additional EDTA-free protease inhibitor-mix tablet (Roche Diagnostics, Mannheim, Germany). Tissue lysates were vortexed and centrifuged to remove unlysed cells and tissue debris. Equal amounts of protein (10 to 25 μg) were run on 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to Immobilon (Millipore, Billerica, MA) membranes. The membranes were blocked, and sequentially probed with a mouse monoclonal antibody to -actin (Sigma Chemical Co, St Louis, MO) as a loading control, mouse monoclonal anti--tubulin clone DM1A (Sigma), mouse monoclonal antiacetylated -tubulin, clone 6-11B-1 (Sigma), and rabbit antidetyrosinated tubulin polyclonal antibody (Chemicon International Inc, Temecula, CA). The membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies, with appropriate species reactivity, and the protein-antibody complexes were detected by using Super-Signal Chemiluminescent reagents (Pierce Biochemicals, Rockford, IL) and quantitated by densitometry.
For immunohistochemistry, formalin-fixed, paraffin-embedded sections were processed as described previously. 16 Acetylated and glu-terminated mouse monoclonal -tubulin antibodies (described above), and mouse monoclonal anti-Ki67 (MIB-1) antibody (Dako, Carpinteria, CA) were applied to tissue sections at a dilution of 1:1000, 1:1000, and 1:50, respectively. The sections were scored quantitatively using the Automated Cellular Imaging System (ACIS; Chromavision, San Juan Capistrano, CA). Six areas of each tumor were scored using a free-scoring or 40x magnification tool to generate an averaged percentage and intensity of stained tumor cells. The staining index was calculated by multiplying the percentage of positively stained cells by the average staining intensity after subtracting the machine readouts of the corresponding negative control for each marker. For Ki67, a labeling percentage was reported.
RESULTS
Patients
In this phase II study, 37 patients were enrolled at the NCI (29 patients) or at the University of Pittsburgh Cancer Institute (eight patients) between June 2002 and March 2004. All patients received at least one cycle and all patients are included in the analysis.
The baseline characteristics of the patients are summarized in Table 1. All patients had at least two cycles of a paclitaxel- or docetaxel-containing regimen (as neoadjuvant, adjuvant, or metastatic therapy) at some point in their therapeutic history. Twenty-two (59%) patients had progressive disease while receiving paclitaxel- and/or docetaxel-containing regimens.
Patients received a median of four cycles (range, one to 11 cycles) of ixabepilone chemotherapy. Of the 37 patients enrolled, 29 patients were removed from study for disease progression, two patients were removed after maximum benefit was achieved (one for neoadjuvant therapy and one for resolution of nonmeasurable disease), five patients were removed for prolonged toxicity, and one patient remains enrolled onto the study.
Efficacy
The objective response was 22% (95% CI, 9.8% to 38.2%), with one complete response and seven partial responses. Stable disease for at least 6 weeks was the best response for 35% of patients (13 patients). The median time to progression for all patients was 80 days (Fig 1). Patients classified as having progressive disease included one patient enrolled and treated with good clinical response but determined to be ineligible retrospectively when found to be without measurable disease by RECIST criteria, one patient with severe toxicity during the first cycle who refused additional evaluation, and two patients who were considered to have progressive disease 8 and 10 days after starting therapy because of life-threatening tumor complications.
For eight patients with confirmed partial responses, the median response duration was 118 days. One patient with a partial response at 6 weeks improved to a confirmed complete response after 8 months (250 days). Measurable responses were seen in lung and liver lesions, and supraclavicular, mediastinal, and axillary lymph nodes. Improvements were also seen in nontarget lesions, such as breast and bone lesions and pleural effusions. Responses were seen in patients who had experienced disease progression after receiving a prior taxane and well as patients previously exposed to an anthracycline, capecitabine, and a taxane (Table 2). One of two patients enrolled who had experienced disease progression after receiving both paclitaxel and docetaxel had a partial response to ixabepilone. One patient with inflammatory breast cancer who had stable disease with neoadjuvant docetaxel and doxorubicin was switched to, and responded to, ixabepilone as a neoadjuvant therapy.
Toxicities
Toxicity information was collected for all patients, who received a total of 153 cycles of therapy. For each patient, the worst grades seen of the most common hematologic and nonhematologic toxicities are summarized in Table 3. One patient was removed from study because of grade 3 sensory neuropathy and one patient was removed from study because of prolonged grade 2 sensory neuropathy. During their first cycle of ixabepilone, two patients had multiple grade 3 and 4 toxicities, including febrile neutropenia, thrombocytopenia, severe mucositis, diarrhea, fatigue, and sensory neuropathy. Neither of these patients received additional treatment with ixabepilone; one of these two patients received concurrent warfarin and clarithromycin, whereas the other did not.
One patient developed slowly progressive patchy pulmonary infiltrates that showed an organizing, nonspecific interstitial pneumonia and metastatic tumor cells on lung biopsy. She was removed from the study, and after receiving high-dose corticosteroids the infiltrate resolved symptomatically and radiologically.
Twelve patients required dose reduction while enrolled onto the study (median, cycle 5; range, cycles 2 to 10). The dose was reduced because of neuropathy in six patients, and because of diarrhea, fatigue, and neutropenia in two patients each. Only one patient required a second dose reduction because of a grade 3 myalgia.
Five patients received erythropoietin or darbepoetin. Four patients received filgrastim or pegfilgrastim for febrile neutropenia. Three patients received prophylactic pegfilgrastim because of prolonged neutrophil recovery in a prior cycle or for a prior episode of febrile neutropenia.
Peripheral Sensory Neuropathy
Of the 24 patients with no baseline neuropathy, 14 patients (58%) developed grade 1, six patients (25%) developed grade 2, and one patient (4%) developed grade 3 peripheral sensory neuropathy as the worst grade of neuropathy. Only two (15%) of 13 patients with baseline grade 1 neuropathy developed grade 2 neuropathy. In total, nine patients developed at least grade 2 neuropathy during the course of their treatment; two of these patients developed grade 2 neuropathy during the first cycle, along with other severe hematologic and nonhematologic toxicities. Of the other seven patients, the median time to development of grade 2 neuropathy was 108 days on study (range, 42 to 189 days). The median times to development and resolution of neuropathy and the outcomes are summarized in Figure 2.
Correlative Studies
Biopsies were collected at baseline and during cycle 2 from five patients (one patient had partial response, two patients had stable disease, and two patients had progressive disease) and were confirmed by pathology to contain tumor. Four of these paired patient biopsies were analyzed by Western blot. For one patient (patient 5), the baseline sample was not adequate. Increased acetylation of -tubulin was seen by Western blot for all four patients (labeled patients 1 through 4 with their responses in Fig 3). Glu-termination was also increased in patients 2, 3, and 4.
Because the protein lysates run on Western blot contain both tumor and stromal components of the biopsy, we next chose to quantify tumor changes alone by immunohistochemistry. Four of the five paired patient biopsies (labeled patients 1, 2, 3, and 5) had tissue available for sectioning for immunohistochemistry. For patient 4, no tumor remained from the baseline paraffin block. For patient 1, who had a partial response to ixabepilone, levels of acetylated -tubulin were detected in both tumor and stromal cells in the baseline sample and increased in both cellular compartments with treatment (Figs 4A and 4B). In contrast, for patient 5, who had progressive disease, levels of acetylated -tubulin were detected only in stromal cells at baseline and increased in the stroma but were still absent in the tumor cells with treatment (Figs 4C and 4D). Baseline levels of acetylated -tubulin were present before treatment and increased after treatment in the patients with response and stable disease (Fig 5A), and levels of glu-terminated -tubulin increased in all patients with treatment (Fig 5B). Ki67 labeling percentage, a measure of tumor proliferation, decreased in the same two patients with increased levels of acetylated -tubulin after treatment, but the Ki67 labeling percentage remained similar in the two patients with no change in the level of acetylated -tubulin after treatment (Fig 5C).
DISCUSSION
Ixabepilone has been studied in a number of phase I and II clinical trials, including one phase I and three phase II breast cancer trials (not including the current study) that have been presented previously in abstract form. Partial response rates for the phase II clinical trials have been 12% for a taxane-resistant population, 17 44% for a population with prior adjuvant or neoadjuvant anthracycline therapy and no prior therapy for metastatic disease, 18 and 31% for another mixed population of breast cancer patients. 19 Because our patients received taxanes in a variety of prior regimens, including neoadjuvant or adjuvant therapy, and the number of prior treatments for metastatic disease varied widely, it is not surprising that our response rate of 22% falls within the range of response rates previously reported. In metastatic breast cancer patients with docetaxel-resistant disease, weekly paclitaxel had a 32% response rate, 20 whereas in patients with paclitaxel-resistant disease, docetaxel had an 18% response rate. 21
In our study, there was a low incidence of hematologic toxicities seen with this schedule, and the most significant complaints were fatigue, myalgia, and peripheral sensory neuropathy. However, for two patients, significant toxicities occurred in the first cycle requiring cessation of additional therapy. In both patients severe febrile neutropenia, nausea, vomiting, diarrhea, mucositis, and peripheral neuropathies occurred within a week of completing the first cycle of treatment. The reason for the comparatively severe toxicities seen in these two patients is unknown. One patient, who had been extensively treated with docetaxel previously without significant complications, received clarithromycin (a cytochrome P450 3A4 inhibitor) on day 4 of cycle 1, leading us to speculate whether a drug-drug interaction may have been responsible for symptoms. However, the second patient did not receive any cytochrome P450 inhibitors. Both patients received warfarin, as did three other patients without severe toxicities. Because blood sampling was not done with this study, pharmacokinetic information regarding drug levels was not available.
One of the most notable complications associated with taxane and other microtubule-targeted treatments is the development of sensory peripheral neuropathy. Although most of our patients who received four or more cycles of ixabepilone had some subjective symptoms of neuropathy, these usually did not interfere with function. Only one patient was considered to have grade 3 neurotoxicity because her peripheral neuropathy was the primary reason she felt compelled to stop working. Grade 3 neurotoxicity was not seen in the phase I study with the regimen administered daily for 5 days. 12 The grade 3 peripheral sensory neuropathy seen with ixabepilone at this schedule (3%) appears to be less than that reported for other ixabepilone schedules (4% to 25%). 19, 22- 26 The lower incidence of neuropathy may be related to the administration schedule, or to the population enrolled onto this study. In our study, all of our patients had received a taxane previously, which may make them more susceptible to either developing a neuropathy or to having a recurrence of previous neurotoxicity. However, because all patients were either considered grade 0 or 1 at study entry, patients who already had a significant unresolved neuropathy would not have been enrolled. For similar reasons, a patient with a history of significant peripheral neuropathy may have been less willing to enroll onto this trial.
the available paired tumor biopsies, we observed increased levels of both glu-terminated and acetylated -tubulin after treatment, indicating that ixabepilone stabilized microtubules in the target tissue. Measures of stabilized microtubules may not only demonstrate a drug effect, but may also help to predict response when examined in a pretreatment specimen. Interestingly, we found that acetylated -tubulin levels were higher at baseline in the tumor cells of patients whose tumors responded or in patients with stable disease than in the patient whose tumor did not respond. In the patient with progressive disease, baseline levels of acetylated -tubulin within the tumor were not detectable by immunohistochemistry. Thus, our results suggest that a tumor with inherent microtubule stability might be more likely to respond to a stabilizing agent with additional microtubule stabilization and decreased proliferation. Although the small numbers of patients biopsied in this group limits our ability to make more definitive statements, these interesting observations are hypothesis generating and should be considered for additional study.
In summary, ixabepilone has shown activity in patients with metastatic breast cancer who have been previously treated with paclitaxel or docetaxel. For most patients, ixabepilone was well tolerated with low hematologic toxicity, and minimal or easily controlled nausea, vomiting, and diarrhea. In addition, administration of ixabepilone does not require premedication with corticosteroids, unlike paclitaxel and docetaxel, which improves its tolerability. Neurotoxicity, a major concern for microtubule-stabilizing drugs, was relatively mild for ixabepilone on this schedule. Its activity and safety profile warrant additional development of this therapy.
Authors' Disclosures of Potential Conflicts of Interest
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Consultant/Advisory Role: Adam Brufsky, Bristol-Myers Squibb. Honoraria: Adam Brufsky, Bristol-Myers Squibb. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Acknowledgment
We thank Diana Nguyen for immunohistochemistry and A. Dimitrios Colevas, MD, for helpful discussions regarding the conduct of this trial.
NOTES
Presented at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, 2004 (preliminary portions of the study, abstract 545).
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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