Allyson Parr

Evaluation of Biologic End Points and Pharmacokinetics in Patients With Metastatic Breast Cancer After Treatment With Erlotinib, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor

PURPOSE To evaluate changes in epidermal growth factor receptor (EGFR) phosphorylation and its downstream signaling in tumor and surrogate tissue biopsies in patients with metastatic breast cancer treated with erlotinib, an EGFR tyrosine kinase inhibitor, and to assess relationships between biomarkers in tumor and normal tissues and between biomarkers and pharmacokinetics. PATIENTS AND METHODS Eighteen patients were treated orally with 150 mg/d of erlotinib. Ki67, EGFR, phosphorylated EGFR (pEGFR), phosphorylated mitogen-activated protein kinase (pMAPK), and phosphorylated AKT (pAKT) in 15 paired tumor, skin, and buccal mucosa biopsies (at baseline and after 1 month of therapy) were examined by immunohistochemistry and analyzed quantitatively. Pharmacokinetic sampling was also obtained. RESULTS The stratum corneum layer and Ki67 in keratinocytes of the epidermis in 15 paired skin biopsies significantly decreased after treatment (P = .0005 and P = .0003, respectively). No significant change in Ki67 was detected in 15 tumors, and no responses were observed. One was EGFR-positive and displayed heterogeneous expression of the receptor, and 14 were EGFR-negative. In the EGFR-positive tumor, pEGFR, pMAPK, and pAKT were reduced after treatment. Paradoxically, pEGFR was increased in EGFR-negative tumors post-treatment (P = .001). Although markers were reduced in surrogate and tumor tissues in the patient with EGFR-positive tumor, no apparent associations were observed in patients with EGFR-negative tumor. CONCLUSION Erlotinib has inhibitory biologic effects on normal surrogate tissues and on an EGFR-positive tumor. The lack of reduced tumor proliferation may be attributed to the heterogeneous expression of receptor in the EGFR-positive patient and absence of target in this cohort of heavily pretreated patients.

Phase I Clinical Trial of Oral 2-Methoxyestradiol, an Antiangiogenic and Apoptotic Agent, in Patients with Solid Tumors

Purpose: To determine the maximum-tolerated dose (MTD) and toxicity profile of the novel anticancer agent, 2-methoxyestradiol (2ME2) administered orally, in patients with solid tumors. Patients and Methods: Twenty patients with refractory solid tumors were enrolled. 2ME2 was given orally starting at 400 mg bid with dose escalation until 3000 mg bid. Tumor biopsies were taken before and after starting the drug to assess for microvessel density by CD 31 and cell proliferation by Ki67 immunohistochemistry. Serial plasma samples collected up to 50 hours after first single oral dose for characterization of pharmacokinetics, were analyzed using liquid chromatography tandem mass-spectrometry. Results: Eleven men and nine women received 2ME2 at dose levels of 400 mg bid (n=3), 800 mg bid (n=3), 1600 mg bid (n=6), 2200 mg bid (n=5) and 3000 mg bid (n=3). There were no dose limiting toxicities, therefore the MTD was not defined. There was one episode of grade 4 angioedema in the 1600 mg bid dose level 38 days into 2ME2 treatment. Other toxicities were mild to moderate. A patient with clear cell carcinoma of the ovary had a partial response at 1600 mg bid dose level lasting over 3 years. Conclusion: MTD for 2ME2 was not reached at dose of 3000 mg bid. The trial was closed due to extremely low plasma concentrations of 2ME2 relative to the doses administered. 2ME2 treatment had no effect on microvessel density (CD31 immunostaining) and cell proliferation (Ki-67 immunostaining). A new formulation of 2ME2 with improved bioavailability is currently being developed.

Phase I trial of the cyclin-dependent kinase inhibitor flavopiridol in combination with Docetaxel in patients with metastatic breast cancer

Purpose: The purpose of this study was to determine the toxicities and characterize the pharmacokinetics of docetaxel and flavopiridol in patients with metastatic breast cancer. Experimental Design: Docetaxel was administered at an initial dose of 60 mg/m2 followed in 24 hours by a 72-hour infusion of flavopiridol at 50 mg/m2/d every 3 weeks. Because dose-limiting myelosuppression occurred, the schedule was amended to docetaxel, 50 mg/m2, followed by escalating doses of flavopiridol (starting dose, 26 mg/m2/d) as a 1-hour infusion daily for 3 days. Pharmacokinetic studies were performed. Ki67, p53, and phosphorylated retinoblastoma protein (phospho-Rb) in paired tumor and buccal mucosa biopsies (obtained pre- and posttreatment) were examined by immunohistochemistry. Results: Eleven patients were enrolled. Five patients received docetaxel and 72-hour flavopiridol. Dose-limiting toxicity was grade 4 neutropenia. Six patients received docetaxel and 1-hour flavopiridol, and the dose-limiting toxicity was grade 3 hypotension. Pharmacokinetics of flavopiridol and docetaxel were consistent with historical data. Nuclear staining with p53 increased and phospho-Rb decreased in 10 pairs of buccal mucosa biopsies posttreatment (P = 0.002 and P = 0.04, respectively). No significant changes in Ki67, p53, or phospho-Rb were detected in six paired tumors. Two patients sustained stable disease for >3 months (72-hour flavopiridol), and one partial response was observed (1-hour flavopiridol). Conclusions: Docetaxel combined with 72-hour flavopiridol was not feasible because of dose-limiting neutropenia. Dose escalation of a 1-hour infusion of flavopiridol with docetaxel was also not possible. The changes in p53 and phospho-Rb in buccal mucosa suggest that a biological effect with flavopiridol was achieved.

5-Fluorouracil-mediated Thymidylate Synthase Induction in Malignant and Nonmalignant Human Cells

Thymidylate synthase (TS, EC 2.1.1.45) is an important target enzyme for the fluoropyrimidines used in cancer chemotherapy. Studies have documented a 2- to 4-fold induction of TS protein following 5-fluorouracil (5-FU) treatment of malignant cells. We measured the effect that 5-FU exposure had on TS protein expression in nonmalignant human breast (MCF-10 and HBL-100), colorectal (ATCC Co18, Co112, and Co33), and bone marrow cells along with malignant breast (MCF-7) and colon (NCI-H630) cells. Twenty-four hours after plating, cells were treated with 0.01 to 10 microM of 5-FU for a period of 24 hr. TS was quantitated by Western immunoblot using monoclonal antibody TS106. Absolute levels of TS in nonmalignant cells were substantially lower than in the malignant lines, ranging from approximately 40% in HBL-100 cells to less than 10% in the colon lines. An approximately two-fold induction in the level of TS was found for all cell lines examined, and there was a strong dependence on 5-FU exposure concentration in free TS levels of MCF-WT, and total TS levels of H630-WT, normal bone marrow, and MCF-10 cells. The induction of TS following 5-FU exposure is a generally observed phenomenon in both malignant and nonmalignant cells, suggesting that a selective means for inhibiting this induction may be critical for the development of alternative therapeutic strategies using 5-FU and the antifolate TS inhibitors.

Thymidylate synthase as a molecular target for drug discovery using the National Cancer Institute's Anticancer Drug Screen.

Thymidylate synthase (TS) is a critical cellular target for cancer chemotherapeutics, particularly the fluoropyrimidine and antifolate classes of antineoplastic agents. One of the primary mechanisms of clinical insensitivity to these agents is through the overexpression of the target enzyme, TS. Thus, there is a need for the development of agents which selectively target TS-overexpressing malignant cells. To this end, we conducted a search for agents which potentially selectively target TS-overexpressing cells using two separate algorithms for identifying such compounds in the NCI Drug Repository by comparing cytotoxicity profiles of 30 000 compounds with the TS expression levels measured by Western blot analysis in 53 cell lines. Using the traditional COMPARE analysis we were unable to identify compounds which maintain a selective ability to kill high TS-expressing cells in a subsequent four cell line validation assay. A new algorithm, termed COMPARE Effect Clusters analysis, enabled the identification of a particular drug cluster which contained compounds that maintained a selective ability to kill TS-overexpressing cell lines in the validation assay. While the identified compounds were selectively cytotoxic to TS-overexpressing cells, we found that they were not specifically targeting TS as a mechanism of action. Apparently, the overexpression of TS was providing a marker for sensitivity. This identified class of compounds which appears to be selectively cytotoxic against cells which overexpress TS may be useful for the development of therapeutics for those whose cancers overexpress TS de novo.

Development of human anti-thymidine kinase antibodies.

Thymidine kinase (TK) is a key enzyme involved in the nucleoside salvage pathway leading to the intracellular synthesis of thymidylate through the phosphorylation of preformed thymidine nucleosides. It has previously been shown that cellular TK levels are regulated by intracellular levels of dTTP. A recent study from our laboratory has shown that exposure to camptothecin analogs, such as 9-aminocamptothecin, results in the inhibition of TK through an indirect mechanism not associated with changes in intracellular dTTP pools. To enable further investigation of this inhibitory mechanism and to provide a reagent useful for other investigations of TK, we have developed anti-TK antibodies in rabbits using peptides of TK. TK is an important determinant of cellular sensitivity to fluoropyrimidines and to several new antifolate inhibitors of thymidylate synthase. Thus, the availability of antibodies against TK would facilitate investigations of the role of this enzyme as a potential predictor of responsiveness to these commonly used chemotherapeutic agents.

Effect of bevacizumab and chemotherapy on serum levels of sVCAM-1 in patient with inflammatory and locally advanced breast cancer

611 Background: Levels of serum vascular cell adhesion molecule (sVCAM-1), a potential surrogate marker for angiogenesis, are elevated in breast cancer. This study aims to assess changes in sVCAM-1 after anti-angiogenic therapy and correlate changes with clinical response.Methods: 21 patients (pts) with inflammatory (IBC) and locally advanced breast cancer (LABC) underwent neoadjuvant treatment with bevacizumab (BV alone) for cycle (C) 1 followed by 6 C of BV and chemotherapy (CTX) with doxorubicin, docetaxel, and G-CSF. Blood samples were collected at baseline (BL), post-BV alone, and post-BV/CTX. sVCAM-1 levels were measured by ELISA. Changes from BL to post treatment levels were evaluated using the Wilcoxon signed rank test. Values at each time point and the two changes from BL were compared between responders and non-responders using an exact Wilcoxon rank sum test. P-values are two-tailed, with a p<0.025 required for significance in view of two primary comparisons being made. Results: There were 14 p...