Clinical Studies with Protein Kinase Inhibitors in Patients with Metastatic Breast Cancer The development of target-based anticancer drugs, such as protein kinase inhibitors, has become an attractive therapeutic strategy in oncology. We are currently studying the role of these compounds, specifically flavopiridol, a cyclin-dependent kinase (CDK) inhibitor, and OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TK), 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 are conducting 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. Eight patients have been enrolled to date. Five patients were enrolled onto the original schedule with flavopiridol as a 72-hour continuous infusion. 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. To date, three patients have enrolled onto the first dose level of the new schedule. At the current time, the maximum tolerated dose of docetaxel in combination with flavopiridol as a 1-hour infusion daily for 3 days has not been determined. The first two patients did not experience any DLT. The third patient was recently treated. If a DLT occurs at this dose level, up to three additional patients must be treated at this dose level before escalation can proceed. If no DLT occurs at this level, then escalation can proceed to the next level. 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. Preliminary immunohistochemical analysis of serial tumor and buccal biopsies revealed a decrease in MIB-1 in three out of the first five patients. There was a decrease in the MIB-1 proliferative index in biopsied samples (tumor and buccal mucosa) from a breast cancer patient with skin involvement post-treatment and a decrease in cyclin D1 expression in her tumor post-treatment. We realize accrual has been less than expected, but note that considerable time was lost in being able to enroll patients because the trial was on hold until an amendment submitted in September 2001 (to change to a 1-hour schedule for flavopiridol) was approved both by the IRB and then by CTEP which took four months (approval was received in December 2001). To increase patient accrual and maintain the timeliness of the study's findings, we have done the following: 1) expanded the population of eligible patients by allowing patients with previous docetaxel treatment to enroll and removed the requirement for patients to have prior paclitaxel and anthracyline exposure and 2) decided not to conduct the phase II portion, but just plan on finishing the phase I part of the study (to determine the maximum tolerated dose and DLT of docetaxel followed by flavopiridol as a 1-hour infusion). OSI-774 OSI-774 is an orally active, potent, and selective inhibitor of the EGFR-TK. Significant growth delay was seen in a range of human tumor xenografts as well as tumor regressions in several tumor models exposed to OSI-774. Breast cancer is associated with aberrant or overexpression of EGFR. Overexpression of EGFR is found in approximately 45% of breast cancers and is associated with disease progression and reduced survival, thus making it an ideal disease for treatment with EGFR-targeted therapy. We are conducting a pilot study to evaluate EGFR signaling after treatment with oral OSI-774 in patients with metastatic breast cancer; the hypothesis is that a reduction in the phosphorylation of EGFR, ERK, and AKT (components of the EGFR pathway) will be detectable in tumor and normal surrogate tissue biopsies of breast cancer patients after exposure to OSI-774. Because not much is known about the relationship between OSI-774's biologic effect in tumor tissue and clinical activity, molecular and pharmacodynamic endpoints (specifically EGFR phosphorylation and downstream indicators of suppression of EGFR function) will be determined. Our primary objectives are to determine whether a change in EGFR phosphorylation is detected in tumors and skin and buccal mucosa (as potential surrogate tissues) before and after treatment with OSI-774, to determine whether a change in other parameters of signal transduction that are downstream of EGFR, namely ERK and AKT, occur in these tissues before and after treatment with OSI-774, and to determine the assay reproducibility of assessing phosphorylation of EGFR, ERK, and AKT via Western blot analysis in biopsied samples before and after treatment with OSI-774. Our secondary objectives are to obtain pharmacokinetic data and attempt to correlate it with a change in EGFR phosphorylation in tumor biopsies, to measure phosphorylated EGFR, ERK, and AKT in tumor tissues via protein microarray analysis, and to evaluate protein binding of OSI-774 via measurement of -1-acid glycoprotein levels. This study opened in December 2001. Our accrual goal is 15 patients. Twelve patients have been enrolled to date. We have successfully obtained biopsy specimens of tumor, normal skin, and normal buccal mucosa pre- and post-treatment on these seven patients. We plan to evaluate all the specimens when accrual is completed. Validation of the antibodies that will be used to analyze the specimens is currently ongoing. This trial will provide an opportunity to directly study the effects of OSI-774 on EGFR-dependent signal transduction pathways functionally before and after treatment to determine whether changes in EGFR phosphorylation and other downstream effectors occur and to determine the assay reproducibility of these markers via Western blots. The latter analysis can give a quantitative assessment of any changes and will complement the immunohistochemical evaluation. Proteomics is an evolving field and tools are being developed that allow the identification of a large number of proteins in biological specimens. This can result in the creation of molecular profiles that may in the future be able to predict a patient's response to therapy. The results of this will also be compared to the Western blot and immunohistochemical analysis. We hope to gain some evidence of a desired biochemical effect and correlate biologic endpoints with OSI-774's therapeutic effect. In summary, we hope that the information obtained from tumor tissue analysis will help to characterize the activity of the drug so that a future trial may be able to define a biological effective dose that may be used in the treatment of early stage breast cancer and DCIS or for chemoprevention. The development of molecularly targeted therapy is an exciting area in the oncology field. The examination of effects of these mechanism-based drugs at the cellular level is a logical approach to test these compounds and may in the future replace the traditional way of determining the appropriate dose of new agents. The development of reproducible assays and tools to assess the effect of a drug on target, such as inhibition of enzyme activity, the validation of surrogate endpoints to check the desired effect of a drug, and the implementation of novel clinical trial designs that allow the testing of biologic endpoints as a primary aim, will become a challenge to both clinicians and basic scientists in the new era of targeted therapy. BMS-247550 BMS-247550 (NSC 710428), an investigational agent, is a semi-synthetic analog of the natural product epothilone B. The epothilones are a novel class of non-taxane microtubule-stabilizing agents obtained from the fermentation of the cellulose degrading myxobacteria, Sorangium cellulosum. BMS-247550 is active against cancer models that are naturally insensitive to paclitaxel or have developed resistance to paclitaxel, both in vitro and in vivo. Metastatic breast cancer is incurable, but often treated palliatively with the taxanes, docetaxel and paclitaxel. While responses are often high, metastatic breast cancer will eventually progress. BMS-247550 may overcome taxane resistance in breast cancer patients previously treated with taxanes. The primary objective of our Phase II Study is to establish the efficacy of BMS-247550 in patients with metastatic breast carcinoma when administered as a one hour infusion on day 1 to 5 every 21 days at 6 mg/m2/day. Patients will be stratified by prior taxane therapy. Secondary objectives are to evaluate mechanisms of drug resistance, specifically by examining tubulin polymerization and p53 expression in tumor samples by immunohistochemistry and profiling gene expression by DNA microarray technology. In addition, for patients enrolled at the NIH Clinical Center, a battery of quantitative exams performed by our Rehabilitation Medicine Department will be assessed as a novel approach to neurotoxicity assessment. These exams are simple, office-based tests that can be performed inexpensively and non-invasively, to assess for early signs of neurotoxicity and functional impairment due to chemotherapy. The proposed sample size is a minimum of 19 evaluable patients and a maximum of 58 total patients, at least 37 evaluable previously taxane-treated patients and 21 evaluable previously untreated patients. This trial began accrual in June 2002 at the NIH Clinical Center. We have also been working cooperatively with the Walter Reed Army Medical Center and the University of Pittsburgh Cancer Institute to begin accrual of patients at those institutions as part of a multicenter trial. As part of this effort, new clinical case report forms have been developed for this trial, and these forms are being used as a model example for the future development of Clinical Center case report forms. National Surgical Adjuvant Breast and bowel Project (NSABP) Adjuvant therapy of breast cancer has clearly provided a significant survival benefit for women in all age groups. Though screening mammography has resulted in earlier detection, still 50 percent of patients with early breast cancer present with node-positive disease. There is a significant recurrence rate in these patients, even with doxorubicin-based chemotherapy. Approaches for increasing the survival include the addition of taxanes. A multicenter trial, NSABP B30, for which Dr. Swain is the protocol chair, is a three-arm trial that will explore the efficacy of docetaxel on survival and quality of life. The three arms include a combination of docetaxel and doxorubicin versus a combination of docetaxel, doxorubicin, and cyclophosphamide, versus doxorubicin and cyclophosphamide followed sequentially by docetaxel. This trial will include 4,000 patients with node-positive breast cancer and has currently accrued 3,300 patients. A quality of life question is being asked in all patients and also specifically in premenopausal women. It has been shown in a meta-analysis of ovarian ablation that this approach increases survival to the same degree as chemotherapy. Therefore, the trial will evaluate ovarian suppression induced by chemotherapy and determine whether this suppression leads to an increased survival. The other quality of life issues relate to the toxicity of the three combinations. Other research areas with NSABP include membership in the Cardiology Advisory Panel for the adjuvant Herceptin trial (NSABP B31) and Protocol Chair for the IressaTM trial. Iressa is an epidermal growth factor tyrosine kinase inhibitor and will be evaluated with docetaxel in metastatic breast cancer. Dr. Swain has found that the hormone receptor status of the contralateral breast cancer (CBC) is markedly associated with that of the primary tumor from analysis of several NSABP trials, 5513 patients. Also, the data is consistent with the hypothesis that tamoxifen treatment reduces the incidence of the ER positive CBC. The histology of the primary was significantly associated with the histology of the CBC. The possibilities for these results include that the CBC could be a metastasis, there is a field effect in all breast tissue making any tumor appear similar in origin, or there is truly a unique second primary.