Tona M. Gilmer

ras Mutations and expression in head and neck squamous cell carcinomas

Mutational activation and overexpression of the family of ras proto‐oncogenes have been associated with many human tumors. The role of mutations of H‐ras, K‐ras, and N‐ras, as well as expression of the respective protein products (p21s) in normal mucosa, dysplastic mucosa, and squamous cell carcinomas (SCCs) of the head and neck has not been fully described. In our study, 51 tumors (40 paraffin embedded and 11 fresh frozen) were examined to determine if mutational activation of ras is an important molecular event in head and neck SCC. Analyses of codons 12, 13, and 61 of H‐ras, K‐ras, and N‐ras revealed no mutations, suggesting that mutational activation of ras is not important in the majority of head and neck SCCs.

Evaluation of Lapatinib and Topotecan Combination Therapy: Tissue Culture, Murine Xenograft, and Phase I Clinical Trial Data

Purpose: Topotecan resistance can result from drug efflux by P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) as well as survival signals initiated by epidermal growth factor receptor family members. The present studies were done to determine the effect of combining topotecan and the dual epidermal growth factor receptor/HER2 inhibitor lapatinib in tissue culture, a murine xenograft model, and a phase I clinical trial. Experimental Design: The effects of lapatinib on topotecan accumulation and cytotoxicity in vitro were examined in paired cell lines lacking or expressing Pgp or BCRP. Antiproliferative effects of the combination were assessed in mice bearing HER2+ BT474 breast cancer xenografts. Based on tolerability in this preclinical model, 37 patients with advanced-stage cancers received escalating doses of lapatinib and topotecan in a phase I trial. Results: Lapatinib increased topotecan accumulation in BCRP- or Pgp-expressing cells in vitro, and the combination showed enhanced efficacy in HER2+ BT474 xenografts. In the phase I study, nausea, vomiting, diarrhea, and fatigue were dose limiting. The maximum tolerated doses were 1,250 mg/d lapatinib by mouth for 21 or 28 days with 3.2 mg/m2 topotecan i.v. on days 1, 8, and 15 of 28-day cycles. Pharmacokinetic analyses showed that combined drug administration resulted in decreased topotecan clearance consistent with transporter-mediated interactions. Seventeen (46%) patients had disease stabilization. Conclusions: The lapatinib/topotecan combination is well tolerated and warrants further study.

Effect of Lapatinib on the Development of Estrogen Receptor–Negative Mammary Tumors in Mice

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

The Ras/p120 GTPase-activating Protein (GAP) Interaction Is Regulated by the p120 GAP Pleckstrin Homology Domain

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter- and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH2-terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intra-molecular binding.

The Translational Research Working Group Developmental Pathway for Anticancer Agents (Drugs or Biologics)

The Translational Research Working Group (TRWG) was created as a national initiative to evaluate the current status of the National Cancer Institutes investment in translational research and envision its future. The TRWG conceptualized translational research as a set of six developmental processes or pathways focused on various clinical goals. One of those pathways describes the development of agents—both small molecules and biologics—for the treatment and prevention of cancer. The Agents Developmental Pathway was conceived not as a comprehensive description of the corresponding real-world processes, but rather as a tool designed to facilitate movement of an agent through the translational process to the point where it can begin definitive clinical testing. This article presents the Agents Developmental Pathway and discusses key challenges associated with the processes described.

Poster 5: Cell Cycle Protein Expression: Potential New Markers in the Tumorigenesis of Head and Neck Squamous Cell Carcinomas

circulating fluorescent-labeled HNSCC cells that have been injected intra-arterially. Two HNSCC cell lines, JHU-020SCC and FaDu, have been used and have been found to adhere to the endothelium of the dorsal skinfold microvasculature. Adherence density of these cells to the vascular endothelium is affected by the prior topical application of the inflammatory cytokine interleukin-1. The dorsal skinfold transparent window chamber model allows analysis of the interactions between human HNSCC cells and the microvasculature in a dynamic system that may be representative of the processes occurring during formation of DM. In addition, the model allows xenotransplantation of human HNSCC into the window chamber, allowing a method for study of tumor growth, angiogenesis, and invasion. (Supported by the Percy Memorial Research Award, American Academy of Otolaryngology-Head and Neck Surgery Foundation, Inc.)