Johnny Gutierrez

Association between HER-2/neu and Vascular Endothelial Growth Factor Expression Predicts Clinical Outcome in Primary Breast Cancer Patients

Purpose: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a) evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b) compare the prognostic significance of VEGF isoforms; and (c) analyze the combined effects of HER-2/neu and VEGF on clinical outcome. Experimental Design: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF121–206 ELISA uses antibodies that bind to VEGF121 and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF165–206 ELISA uses antibodies that bind to VEGF165 and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF121–206 and VEGF165–206 were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Results: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF121–206 and VEGF165–206 expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (χ2 test: P < 0.001 for both VEGF121–206 and VEGF165–206). VEGF121–206 and VEGF165–206 demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF121–206, P = 0.0068; VEGF165–206, P = 0.0046; multivariate analysis: VEGF121–206, P = 0.1475; VEGF165–206, P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF121–206 and VEGF165–206 were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF121–206, P = 0.0003; VEGF165–206, P = 0.0038; multivariate analysis: VEGF121–206, P = 0.0103; VEGF165–206, P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF121–206, P = 0.0280; VEGF165–206, P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF121–206/VEGF165–206 results in additional prognostic information for survival (VEGF121–206, P = 0.0133; VEGF165–206, P = 0.0092). Conclusion: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

TMEFF2 is a PDGF-AA binding protein with methylation-associated gene silencing in multiple cancer types including glioma.

Background TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers. Methodology/Principal Findings Here we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA–stimulated fibroblast proliferation in a dose–dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype. Conclusions/Significance These data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers.

Pharmacokinetics and pharmacodynamics of DSTA4637A: A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus in mice

ABSTRACT DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.

A New ELISA for Use in a 3-ELISA System to Assess Concentrations of VEGF Splice Variants and VEGF110 in Ovarian Cancer Tumors

BACKGROUND Vascular endothelial growth factor (VEGF), which affects tumor angiogenesis, is expressed as different splice variants, including the major isoforms VEGF(165) and VEGF(121), and can be cleaved by plasmin to generate VEGF(110). The amount of VEGF(121) and VEGF(110) in biological samples has not been well studied. METHODS We developed an ELISA that detects VEGF(165) and VEGF(121) equally, but does not detect VEGF(110). We used this ELISA together with 2 other ELISAs, one detecting VEGF(165) and the other detecting VEGF(165), VEGF(121), and VEGF(110) equally, to assess the concentrations of VEGF(121) and VEGF(110) in ovarian cancer tumors. RESULTS The median concentrations in ovarian cancer tumor lysates were 0.61 (range <0.055-74) fmol/mg protein for VEGF(165), 1.4 (range <0.20-500) fmol/mg protein for VEGF(165) plus VEGF(121), and 2.3 (range <0.079-520) fmol/mg protein for total VEGF including VEGF(110) (n = 248). VEGF concentrations measured by the 3 ELISAs were highly correlated (r = 0.91-0.94). Median estimated VEGF(121) and VEGF(110) concentrations were 0.77 and 0.58 fmol/mg protein, respectively. In lysates with measurable VEGF(165) and total VEGF concentrations, mean VEGF(165) was approximately 31% (SD 23%) of the total VEGF (n = 217). In contrast, VEGF(165) constituted approximately half of the total circulating VEGF. CONCLUSION VEGF(165), VEGF(121), and VEGF(110) may be present at significant amounts in ovarian cancer tumors.

A Prognostic Model of Persistent Bacteremia and Mortality in Complicated Staphylococcus aureus Bloodstream Infection

BACKGROUND Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies. METHODS To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124). RESULTS We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012). CONCLUSIONS These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.