Robert Radinsky

Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer

PURPOSE Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials. PATIENTS AND METHODS KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status. RESULTS KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population. CONCLUSION Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.

Wild-Type Human p53 and a Temperature-Sensitive Mutant Induce Fas/APO-1 Expression

Fas/APO-1 is a cell surface protein known to trigger apoptosis upon specific antibody engagement. Because wild-type p53 can activate transcription as well as induce apoptosis, we queried whether p53 might upregulate Fas/APO-1. To explore this possibility, we examined human p53-null (H358 non-small-cell lung adenocarcinoma and K562 erythroleukemia) and wild-type p53-containing (H460 non-small-cell lung adenocarcinoma) cell lines. When H358 or H460 cells were transduced with a replication-deficient adenovirus expression construct containing the human wild-type p53 gene but not with vector alone, a marked upregulation (approximately a three-to fourfold increase) of cell surface Fas/APO-1 was observed by flow cytometry. Similarly, K562, cells stably transfected with a plasmid vector containing the temperature-sensitive human p53 mutant Ala-143 demonstrated a four- to sixfold upregulation of Fas/APO-1 by flow-cytometric analysis at the permissive temperature of 32.5 degrees C. Temperature-sensitive upregulation of Fas/APO-1 in K562 Ala-143 cells was verified by immunoprecipitation and demonstrated to result from enhanced mRNA production by nuclear run-on and Northern (RNA) analyses. Stably transfected K562 cells expressing temperature-insensitive, transcriptionally inactive p53 mutants (His-175, Trp-248, His-273, or Gly-281) failed to upregulate Fas/APO-1 at either 32.5 degrees or 37.5 degrees C. The temperature-sensitive transcription of Fas/APO-1 occurred in the presence of cycloheximide, indicating that de novo protein synthesis was not required and suggested a direct involvement of p53. Collectively, these observations argue that Fas/APO-1 is a target gene for transcriptional activation by p53.

Orthotopic models are necessary to predict therapy of transplantable tumors in mice.

Rapid evaluation of new cytotoxic agents and biological response modifiers for therapy of cancer and elucidation of their mechanisms of action require the use of relevant animal models. It is well established that the faithful reproduction of the tumor microenvironment that allows the emergence of subpopulations of tumor cells with the biological and metastatic properties observed in clinical cancer occurs with orthotopic tumor models (transplantable and transgenic). This review summarizes the evidence that phenotypic properties of metastatic cells are governed by the expression of genes that are regulated by interaction with the relevant organ environment. While ectopic models of cancer allow rapid screening of new compounds and transgenic models afford opportunities to study early cellular and molecular events in tumor progression and metastasis, orthotopic transplantation of tumor cells remains an affordable, reproducible and reliable methodology for the study of organ-specific determinants of the biology and therapy of cancer.

Fully Human Monoclonal Antibodies to Hepatocyte Growth Factor with Therapeutic Potential against Hepatocyte Growth Factor/c-Met–Dependent Human Tumors

c-Met is a well-characterized receptor tyrosine kinase for hepatocyte growth factor (HGF). Compelling evidence from studies in human tumors and both cellular and animal tumor models indicates that signaling through the HGF/c-Met pathway mediates a plethora of normal cellular activities, including proliferation, survival, migration, and invasion, that are at the root of cancer cell dysregulation, tumorigenesis, and tumor metastasis. Inhibiting HGF-mediated signaling may provide a novel therapeutic approach for treating patients with a broad spectrum of human tumors. Toward this goal, we generated and characterized five different fully human monoclonal antibodies that bound to and neutralized human HGF. Antibodies with subnanomolar affinities for HGF blocked binding of human HGF to c-Met and inhibited HGF-mediated c-Met phosphorylation, cell proliferation, survival, and invasion. Using a series of human-mouse chimeric HGF proteins, we showed that the neutralizing antibodies bind to a unique epitope in the beta-chain of human HGF. Importantly, these antibodies inhibited HGF-dependent autocrine-driven tumor growth and caused significant regression of established U-87 MG tumor xenografts. Treatment with anti-HGF antibody rapidly inhibited tumor cell proliferation and significantly increased the proportion of apoptotic U-87 MG tumor cells in vivo. These results suggest that an antibody to an epitope in the beta-chain of HGF has potential as a novel therapeutic agent for treating patients with HGF-dependent tumors.

AMG 706, an Oral, Multikinase Inhibitor that Selectively Targets Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors, Potently Inhibits Angiogenesis and Induces Regression in Tumor Xenografts

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706-treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies.

Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth, metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice

Vascular endothelial growth factor (VEGF) is the major pro‐angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor‐2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor‐2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti‐VEGF receptor‐2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti‐VEGF receptor‐2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.

Impact of Insulin-Like Growth Factor Receptor-I Function on Angiogenesis, Growth, and Metastasis of Colon Cancer

Insulin-like growth factors and their principal receptor, IGF-I receptor (IGF-IR), are frequently expressed in human colon cancers and play a role in preventing apoptosis, enhancing cell proliferation, and inducing expression of vascular endothelial growth factor (VEGF). The role of IGF-IR in regulating angiogenesis and metastases of human colon cancer has not been elucidated. To determine the in vitro and in vivo effects of IGF-IR in human colon cancer growth and angiogenesis, human KM12L4 colon cancer cells were transfected with a truncated dominant-negative form of IGF-IR (IGF-IR dom-neg). IGF-IR dom-neg–transfected cells demonstrated markedly decreased constitutive expression of VEGF mRNA and protein. Subcutaneous injections of IGF-IR dom-neg–transfected cells in nude mice led to significantly decreased tumor growth (p < 0.05) that was associated with decreased tumor cell proliferation, VEGF expression, and vessel count and with increased tumor cell apoptosis (p < 0.05 for all parameters compared with controls). In addition, pericyte coverage of endothelial cells was significantly decreased in tumors from IGF-IR dom-neg–transfected cells. Following this observation, we demonstrated in vitro that vascular smooth muscle cells migrated significantly less in conditioned medium derived from IGF-IR dom-neg–transfected cells compared with medium from control cells. After splenic injections, IGF-IR dom-neg transfectants failed to produce liver metastases, in contrast to parental cells and mock transfectants (p < 0.05). In addition, IGF-IR dom-neg–transfected cells failed to form liver tumors after direct injection into the liver. These studies demonstrate that the IGF-IR plays an important role in multiple mechanisms that mediate the growth, angiogenesis, and metastasis of human colon cancer. IGF-IR is a valid target for the therapy of human colon cancer.

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.

Association of K-ras Mutational Status and Clinical Outcomes in Patients with Metastatic Colorectal Cancer Receiving Panitumumab Alone

BACKGROUND Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CA gene mutations with tumor resistance to panitumumab alone. PATIENTS AND METHODS From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing. RESULTS Of the 62 samples, 24 (38.7%) harbored a K-ras mutation, and 38 (61.3%) were wild type. In the wild-type K-ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K-ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K-ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K-ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation. CONCLUSION These data suggest that patients with mCRC with activating K-ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.

AMG 102, A Fully Human Anti-Hepatocyte Growth Factor/Scatter Factor Neutralizing Antibody, Enhances the Efficacy of Temozolomide or Docetaxel in U-87 MG Cells and Xenografts

Purpose: Hepatocyte growth factor (HGF/SF) and its receptor c-Met have previously been shown to be up-regulated in multiple human cancers, including glioblastoma multiforme. To better understand if AMG 102, a fully human, anti-HGF/SF–neutralizing antibody, could be incorporated into current clinical practice, AMG 102 was tested preclinically in combination with temozolomide or docetaxel to determine if enhanced efficacy was observed compared with AMG 102 alone. Experimental Design: The effects of AMG 102 were tested for antiproliferative activity in combination with temozolomide or docetaxel on U-87 MG cells in vitro and for antitumor activity in a U-87 MG xenograft model in vivo. Apoptotic activity was also measured for AMG 102 and docetaxel combined in vitro. Results: Treatment with temozolomide combined with AMG 102 resulted in increased inhibition of cell growth in vitro compared with treatment with either single agent alone. In U-87 MG xenografts in vivo, AMG 102 combined with temozolomide or docetaxel significantly increased the inhibitory effect on tumor growth when compared with treatment with either agent alone (P < 0.0001 and P < 0.015, respectively). In vitro, docetaxel alone induced both caspase-3/7 activity as well as poly(ADP)ribose polymerase and caspase-7 cleavage in U-87 MG cells; these events were enhanced when used in combination with AMG 102. Importantly, there was no evidence of interference between AMG 102 and either temozolomide or docetaxel in vitro or in vivo. Conclusion: These studies support testing of AMG 102 in combination with temozolomide or docetaxel. Such combinations may represent promising, novel clinical therapeutic strategies for cancers that are dependent on the HGF/SF/SF:c-Met pathway in the oncology setting.