Studies in Early and Metastatic Breast Cancer The development of target-based anticancer drugs, such as protein kinase inhibitors, has become an attractive therapeutic strategy in oncology. We studied the role of several compounds, specifically flavopiridol (cyclin-dependent kinase CDK) inhibitor, erlotinib (epidermal growth factor receptor tyrosine kinase inhibitor EGFR-TK, and ixabepilone in the treatment of patients with metastatic breast cancer. Flavopiridol Flavopiridol is a synthetic flavone derived from rohitukine, a natural product isolated from a plant indigenous to India. Flavopiridol became the first CDK inhibitor to be tested in clinical trial and the first phase I study was performed at the NCI. There is preclinical evidence that flavopiridol has potent effects in vitro and antitumor activity in vivo in breast cancer. Several in vitro studies also demonstrate flavopiridol's ability to enhance apoptosis induced by chemotherapy, including taxanes, which are agents active against breast cancer. We conducted a phase I/II study of docetaxel followed by flavopiridol in patients with metastatic breast cancer. Our hypothesis is that the addition of flavopiridol to docetaxel will increase the objective response rate in the treatment of patients with breast cancer. Our primary objective is to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of docetaxel followed by flavopiridol. Secondary objectives include the determination of the activity as measured by objective response rate of the combination and the evaluation of cell cycle parameters in tumor and buccal mucosa biopsies before and after treatment with the combination. This study opened in September 2000 and closed in January 2003. Five patients were enrolled onto the original schedule with flavopiridol as a 72-hour continuous infusion. Docetaxel was administered as a 1-hour infusion at an initial dose of 60 mg/m2 followed in 24 hours by a 72-hour infusion of flavopiridol at 50 mg/m2/day every 3 weeks. Because three of these patients experienced myelosuppression (grade 4 neutropenia for 7 days), the study was amended to administer flavopiridol as a 1-hour infusion and to start at a lower dose of docetaxel. Six patients were enrolled onto the new schedule. The purpose of this study was to determine the maximum-tolerated dose and dose-limiting toxicities of docetaxel followed by flavopiridol in patients with previously treated metastatic breast cancer. The efffect of docetaxel and flavopiridol on Ki67, (phosphorylated retinoblastoma protein) Rb, and p53 was explored. A total of eleven patients with metastatic breast cancer were enrolled. Five patients received docetaxel followed by flavopiridol as a 72-hour continuous infusion. Three of these patients experienced dose-limiting toxicity of grade 4 neurotropenia. The other six patients received docetaxel followed by flavopiridol as a 1-hour infusion daily for 3 days. On this schedule, dose-limiting toxicity was grade 3 hypotension. There were two patients that sustained stable disease (72-hour flavopirodol) and one partial response (1-hour flavopiridol). The immunohistochemical evaluation for cyclin D1, p53, ki67/MIB1, bcl2, and apoptosis by TUNEL assay of paired tumor (when available) and buccal mucosa biopsy samples is being performed by the Laboratory of Pathology and with Dr. Xiaowei Yang in the Molecular Profiling Core. Flavopiridol is the first cyclin dependent kinase (cdk) inhibitor to be tested in clinical trials. It is a synthetic flavone derived from rohitukine, a natural product from a plant indigenous to India. Flavopiridol potently competes with ATP to inhibit several cdks, including cdk1, cdk2, and cdk4, with IC50 values in the range of 100-400 nmol/L. Flavopiridol has been shown to arrest breast cancer cells at the G1/S and G2/M transitions. In preclinical models, flavopiridol is cytostatic when given over a protracted schedule-an observation that formed the basis for initiating the phase I clinical trials with a 72-hour continuous infusion. Two phase I trials were completed at the NCI. In vitro, flavopiridol enhanced apoptosis induced by chemotherapy, including taxanes, which are active agents against breast cancer. Preclinical studies of flavopiridol with paclitaxel demonstrated a sequence-dependent induction of apoptosis, in which flavopiridol must be given after paclitaxel to achieve the effect.On the basis of the preclinical sequence-dependent activity, we conducted a phase I study of docetaxel followed by a 72-hour infusion of flavopiridol every 3 weeks. We hypothesized that the addition of a cdk inhibitor that prevents progression through the cell cycle after treatment with a microtubule-stabilizing agent that acts in the mitosis or M phase of the cell cycle would increase the efficacy of the taxane. When dose-limiting myelosuppression occurred, the protocol was amended to administer flavopiridol as a 1-hour infusion daily for 3 days every 3 weeks. Neither changing the schedule nor lowering the dose of flavopiridol altered the toxicity profile. Because severe toxicity made completing this trial difficult and because severe toxicity was noted in another trial that used a 24-hour infusion, we did not to proceed to a phase II study. Translational endpoints from the phase I study provided insight into the activity of the drug in buccal mucosa, as a surrogate, and tumor tissue. We hypothesized that treatment of patients with flavopiridol would decrease the amount of phosphorylated retinoblastoma protein (phospho-Rb) and decrease proliferation, as measured by Ki67 expression. Rb is the major substrate that undergoes phosphorylation by the cyclin D/CDK4 or CDK6 unit, which is essential to move the cell from G1 to S phase. Preclinical studies in breast cancer cells had shown that levels of Rb phosphorylation declined by 12 hours after treatment with flavopiridol. Furthermore, it has been shown that hypophosphorylated Rb inhibits MDM2 and promotes the release of active p53. Therefore, we hypothesized that flavopiridol would increase the expression of p53, a protein with diverse functions related to proliferation, survival, and apoptosis, and evaluated p53 protein expression in serial biopsies collected during the trial. There are no published reports of Ki67, phospho-Rb, or p53 expression in tissue specimens collected after in vivo treatment with flavopiridol. We examined expression of Ki67, p53, and phospho-Rb (as a marker of cdk inhibition) in breast cancer tumor specimens and buccal mucosa (as a readily accessible surrogate tissue) before and after treatment with flavopiridol. In 10 paired buccal biopsies, the mean (? SD) pre-treatment level for Ki67 was 13.6% ? 1.9% and the post-treatment level was 12.5% ? 2.1% (P = 0.71). There was a reduction in the level of phospho-Rb in buccal mucosa samples (P = 0.04). The mean pre-treatment level for p53 was 2.8% ? 1.0% and the post-treatment level was 9.8% ? 2.0% (P = 0.002). A similar trend (decrease in Ki67, increase in p53) was seen in the 6 paired tumor biopsies. For Ki67, the mean pre-treatment level was 29.7% ? 7.6% and the post-treatment level was 23.8% ? 7.2% (P = 0.31). For p53, the mean pre-treatment level was 8.3% ? 5.4% and the mean post-treatment level 14.2 ? 6.3% (P = 0.09). The increase in p53 was seen in 5 of 6 (83%) paired tumor samples. There was no significant change in phospho-Rb expression in paired tumor samples (P = 0.50). The significant increase in p53 expression in buccal mucosa could be the result of the action of flavopiridol binding to DNA or the result of an inhibition of transcription and downregulation of the MDM2 protein, which has been reported to occur with other cdk inhibitors. The decrease in phospho-Rb post-treatment in buccal mucosa suggests that cdk4 kinase activity was inhibited by flavopirido.
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