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.

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments.

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

Opportunities and Challenges in Implementation of Multiparameter Single Cell Analysis Platforms for Clinical Translation.

The high-content interrogation of single cells with platforms optimized for the multiparameter characterization of cells in liquid and solid biopsy samples can enable characterization of heterogeneous populations of cells ex vivo. Doing so will advance the diagnosis, prognosis, and treatment of cancer and other diseases. However, it is important to understand the unique issues in resolving heterogeneity and variability at the single cell level before navigating the validation and regulatory requirements in order for these technologies to impact patient care. Since 2013, leading experts representing industry, academia, and government have been brought together as part of the Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium to foster the potential of high-content data integration for clinical translation.

Inhibition of anchorage-independent growth of transformed NIH3T3 cells by epithelial protein lost in neoplasm (EPLIN) requires localization of EPLIN to actin cytoskeleton.

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein characterized by the presence of a single centrally located lin-11, isl-1, and mec-3 (LIM) domain. We have reported previously that EPLIN is down-regulated in transformed cells. In this study, we have investigated whether ectopic expression of EPLIN affects transformation. In untransformed NIH3T3 cells, retroviral-mediated transduction of EPLIN did not alter the cell morphology or growth. NIH3T3 cells expressing EPLIN, however, failed to form colonies when transformed by the activated Cdc42 or the chimeric nuclear oncogene EWS/Fli-1. This suppression of anchorage-independent growth was not universal because EPLIN failed to inhibit the colony formation of Ras-transformed cells. Interestingly, the localization of EPLIN to the actin cytoskeleton was maintained in the EWS/Fli-1- or Cdc42-transformed cells, but not in Ras-transformed cells where it was distributed heterogeneously in the cytoplasm. Using truncated EPLIN constructs, we demonstrated that the NH(2)-terminal region of EPLIN is necessary for both the localization of EPLIN to the actin cytoskeleton and suppression of anchorage-independent growth of EWS/Fli-1-transformed cells. The LIM domain or the COOH-terminal region of EPLIN could be deleted without affecting its cytoskeletal localization or ability to suppress anchorage-dependent growth. Our study indicates EPLIN may function in growth control by associating with and regulating the actin cytoskeleton.

Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility.

Periostin (PN) is a secreted protein that shares a structural homology to the axon guidance protein fasciclin I in insects. Previously, we reported that PN expression is up-regulated in epithelial ovarian tumors. We further examined the role of PN in ovarian cancer. PN is expressed in several normal tissues but not in normal ovaries and has a tendency for higher expression in fetal tissues. Ovarian cancer cells secrete PN, which can accumulate in malignant ascites of ovarian cancer patients. Purified recombinant PN supports adhesion of ovarian epithelial cells that can be inhibited by monoclonal antibodies against alpha(V)beta(3) or alpha(V)beta(5) integrin, but not by anti-beta(1) integrin antibody. Furthermore, alpha(V)beta(3) integrin, but not beta(1) integrins, colocalizes to the focal adhesion plaques formed on PN. Cells plated on PN form fewer stress fibers and are more motile compared with those plated on fibronectin. We propose PN functions as a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins to support adhesion and migration of ovarian epithelial cells.

Gene expression in epithelial ovarian carcinoma.

We analysed the mRNA levels corresponding to 12,600 transcripts in primary cultures of ovarian epithelial cells derived from nine normal ovaries and 21 epithelial ovarian carcinoma. The class distinction and hierarchical clustering of expression data revealed a clear distinction in gene expression between normal and carcinoma-derived ovarian epithelial cells. Comparison of expression levels revealed 111 genes with mean expression values of >2.5-fold higher in carcinoma cells. Similarly, 62 genes were expressed at >2.5-fold higher levels in normal ovarian epithelial cells. For a few selected genes, we demonstrate that the pattern of differential expression observed in cultured epithelial cells is present in the normal ovaries and epithelial ovarian carcinoma. Use of cultured epithelial cells represents a novel strategy to study gene expression in a cell-type specific manner.

Imatinib mesylate (Gleevec) inhibits ovarian cancer cell growth through a mechanism dependent on platelet-derived growth factor receptor alpha and Akt inactivation.

PURPOSE: We identified the platelet-derived growth factor receptor alpha (PDGFRalpha) as an ovarian cancer-specific gene by microarray hybridization using primary cultures. The purpose of this study is to evaluate whether disruption of the platelet-derived growth factor-regulated growth pathway by Imatinib mesylate (Gleevec), a partially selective PDGFR inhibitor, inhibits growth of ovarian cancer cells expressing PDGFR. EXPERIMENTAL DESIGN: To investigate the effects of Imatinib mesylate in ovarian cancer, we established an in vitro model by immortalizing primary ovarian cells, which express endogenous PDGFR, and we evaluated the effects of Imatinib on cell proliferation. In addition, we investigated the involvement of Akt in mediating Imatinib-inhibited cell growth inhibition. RESULTS: We found that 39% of ovarian tumors express PDGFR by immunohistochemistry. We showed that Imatinib inhibits the growth of ovarian cancer cells in a PDGFR-specific manner, at clinically relevant concentrations (IC(50) < 1 micro M). Imatinib inhibits the growth of three primary ovarian cultures and two immortalized cultures (PDGFR positive), but has no effects on SkOv3 and CaOv3 cell lines (PDGFR negative). Imatinib exerts antiproliferative effects by arresting cells at G(0)-G(1) and preventing progression through S phase. Imatinib inhibits both PDGFRalpha and Akt phosphorylation at a concentration of 1 micro M. Stable expression of constitutively active Akt induces partial resistance to PDGFR inhibition in ovarian cancer cells, as demonstrated by cell proliferation assay and cell cycle analysis. CONCLUSIONS: Our data indicate that Imatinib mesylate inhibits the growth of ovarian cancer cells through PDGFR inactivation. In addition, our results suggest that constitutive Akt activation modulates sensitivity to Imatinib in ovarian cancer cells.

Neutralizing monoclonal antibody to periostin inhibits ovarian tumor growth and metastasis.

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein.

Optimal timing of coronary artery bypass after acute myocardial infarction: a review of California discharge data.

OBJECTIVE: The optimal timing for coronary artery bypass grafting after acute myocardial infarction is not well established. The California Discharge Database facilitates the study of this issue by providing data from a large patient cohort free of institutional bias. We examine the timing of coronary artery bypass grafting after acute myocardial infarction on short-term outcomes. METHODS: We reviewed California Discharge Data to identify 40,159 patients who were hospitalized for acute myocardial infarction (day 0) and underwent subsequent coronary artery bypass grafting. Patients were stratified by the timing of coronary artery bypass grafting to "early" (days 0-2) and "late" groups (day 3 or later). The primary outcome variable was all-cause hospital mortality. Multiple logistic and linear regression and propensity analyses assessed the risk of adverse events, controlling for factors associated with preoperative clinical acuity, including the Charlson Comorbidity Index, shock, mechanical ventilation, and the use of intra-aortic balloon counterpulsation. RESULTS: Of 9476 patients identified, 4676 (49%) were in the early coronary artery bypass grafting group and 4800 (51%) were in the late coronary artery bypass grafting group. A total of 444 patients (4.7%) died during hospitalization, with a peak mortality rate of 8.2% among patients undergoing coronary artery bypass grafting on day 0, declining to a nadir of 3.0% among patients undergoing coronary artery bypass grafting on day 3. The mean time to coronary artery bypass grafting was 3.2 days. Patients undergoing early coronary artery bypass grafting experienced a higher mortality rate than those undergoing late coronary artery bypass grafting (5.6% vs 3.8%, P < .001). Early coronary artery bypass grafting was an independent predictor of mortality after controlling for clinical acuity and on propensity analysis (odds ratio 1.43, P = .003). CONCLUSION: Patients undergoing coronary artery bypass grafting within 2 days of hospitalization for acute myocardial infarction experienced higher mortality rates than those undergoing coronary artery bypass grafting 3 or more days after acute myocardial infarction, independently of clinical acuity. This suggests that coronary artery bypass grafting may best be deferred for 3 or more days after admission for acute myocardial infarction in nonurgent cases.

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.

Safety, pharmacokinetics, and efficacy of AMG 706, an oral multikinase inhibitor, in patients with advanced solid tumors.

PURPOSE: AMG 706 is an investigational, orally bioavailable inhibitor of vascular endothelial growth factor receptors 1, 2, and 3, platelet-derived growth factor receptor, and stem-cell factor receptor. This phase I, dose-finding study evaluated the safety, pharmacokinetics, and pharmacodynamics of AMG 706 in patients with refractory advanced solid tumors. PATIENTS AND METHODS: AMG 706 was administered at escalating doses of 50 to 175 mg once daily or 25 mg bid for the first 21 days of a 28-day cycle. The 125-mg once-daily dose was also administered continuously. The maximum-tolerated dose (MTD), safety, pharmacokinetics, tumor response, and serum levels of proangiogenic markers were determined. RESULTS: Seventy-one patients received AMG 706. The MTD was 125 mg once daily administered continuously. The most frequent adverse events were fatigue (55%), diarrhea (51%), nausea (44%), and hypertension (42%). Plasma AMG 706 concentrations increased in a dose-proportional manner with no accumulation after multiple doses. Five patients (7%) had a partial response, 35 patients (49%) had stable disease (at least through day 50), and 31 patients (44%) had progressive disease. Changes in tumor size correlated significantly with an increase in placental growth factor (P = .003) and a decrease in soluble kinase domain receptor (P = .001). CONCLUSION: In this study of patients with advanced refractory solid tumors, AMG 706 was well tolerated and displayed favorable pharmacokinetics and evidence of antitumor activity. Additional studies of AMG 706 as monotherapy and in combination with various agents are ongoing.

ROCK-II-induced membrane blebbing and chromatin condensation require actin cytoskeleton.

Ectopic expression of ROCK II (Rho kinase II or ROKalpha), an effector of Rho GTPase, induces membrane blebbing and chromatin condensation. ROCK II can induce membrane blebbing in the presence of the caspase inhibitor z-VAD-fmk or in caspase-3-deficient MCF-7 cells, indicating that the activation of caspases is not required. ROCK-II-induced membrane blebbing, however, is reversed by the myosin light chain kinase inhibitor ML-7 or cytochalasin D. In addition, the expression of a constitutively activated form of cofilin (S3A-cofilin) suppresses both membrane blebbing and chromatin condensation in ROCK II expressing cells. These findings suggest that the activation of actin-myosin contractility is responsible for membrane blebbing and chromatin condensation and implicate ROCK II as a potential mediator of the morphological changes associated with apoptosis.

Molecular basis for interaction between Icap1 alpha PTB domain and beta 1 integrin.

Icap1 alpha is a 200-amino acid protein that binds to the COOH-terminal 13 amino acids ((786)AVTTVVNPKYEGK(798)) of the integrin beta(1) subunit. Alanine scanning mutagenesis of this region revealed that Val(787), Val(790), and (792)NPKY(795) are critical for Icap1 alpha binding. The NPXY motif is a known binding substrate for phosphotyrosine binding (PTB) domain proteins. The sequences of Icap1 alpha, residues 58--200, and the beta(1) integrin, residues 786-797, were aligned to the available PTB-peptide structures to generate a high quality structural model. Site-directed mutagenesis showed that Leu(135), Ile(138), and Ile(139) of Icap1 alpha, residues predicted by the model to be in close proximity to (792)NPKY(795), and Leu(82) and Tyr(144), residues expected to form a hydrophobic pocket near Val(787), are required for the Icap1 alpha-beta(1) integrin interaction. These findings indicate that Icap1 alpha is a PTB domain protein, which recognizes the NPXY motif of beta(1) integrin. Furthermore, our date suggest that an interaction between Val(787) and the hydrophobic pocket created by Leu(82) and Tyr(144) of Icap1 alpha forms the basis for the specificity of Icap1 alpha for the beta(1) integrin subunit.