Jason M. Warram

Safety and Tumor-specificity of Cetuximab-IRDye800 for Surgical Navigation in Head and Neck Cancer

Purpose: Positive margins dominate clinical outcomes after surgical resections in most solid cancer types, including head and neck squamous cell carcinoma. Unfortunately, surgeons remove cancer in the same manner they have for a century with complete dependence on subjective tissue changes to identify cancer in the operating room. To effect change, we hypothesize that EGFR can be targeted for safe and specific real-time localization of cancer. Experimental Design: A dose escalation study of cetuximab conjugated to IRDye800 was performed in patients (n = 12) undergoing surgical resection of squamous cell carcinoma arising in the head and neck. Safety and pharmacokinetic data were obtained out to 30 days after infusion. Multi-instrument fluorescence imaging was performed in the operating room and in surgical pathology. Results: There were no grade 2 or higher adverse events attributable to cetuximab-IRDye800. Fluorescence imaging with an intraoperative, wide-field device successfully differentiated tumor from normal tissue during resection with an average tumor-to-background ratio of 5.2 in the highest dose range. Optical imaging identified opportunity for more precise identification of tumor during the surgical procedure and during the pathologic analysis of tissues ex vivo. Fluorescence levels positively correlated with EGFR levels. Conclusions: We demonstrate for the first time that commercially available antibodies can be fluorescently labeled and safely administered to humans to identify cancer with sub-millimeter resolution, which has the potential to improve outcomes in clinical oncology. Clin Cancer Res; 21(16); 3658–66. ©2015 AACR.

A Triple-Targeted Ultrasound Contrast Agent Provides Improved Localization to Tumor Vasculature

Actively targeting ultrasound contrast agents to tumor vasculature improves contrast‐enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single‐, dual‐, and triple‐targeted motifs.

Breast Tumor Xenografts: Diffusion-weighted MR Imaging to Assess Early Therapy with Novel Apoptosis-Inducing Anti-DR5 Antibody

PURPOSE To measure the early therapeutic response to a novel apoptosis-inducing antibody, TRA-8, by using diffusion-weighted magnetic resonance (MR) imaging in a mouse breast cancer model. MATERIALS AND METHODS Animal experiments had institutional animal care and use committee approval. Four groups of nude mice bearing luciferase-positive breast tumors (four to five mice with eight to 10 tumors per group) were injected intravenously with 0 mg (group 1), 0.025 mg (group 2), 0.100 mg (group 3), or 0.200 mg (group 4) of TRA-8 on days 0 and 3. Diffusion-weighted imaging, anatomic MR imaging, and bioluminescence imaging were performed on days 0, 3, and 6 before dosing. Averaged apparent diffusion coefficients (ADCs) for both whole tumor volume and a 1-mm peripheral tumor shell were calculated and were compared with tumor volume and living tumor cell changes. After imaging at day 6, proliferating and apoptotic cell densities were measured with Ki67 and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling, or TUNEL, staining, respectively, and were compared with cleaved caspase-3 density. RESULTS The ADC increase at day 3 was dependent on TRA-8 dose level, averaging 6% +/- 3 (standard error of mean), 19% +/- 4, 14% +/- 4, and 34% +/- 7 in the whole tumor volume and 1% +/- 2, 9% +/- 5, 13% +/- 5, and 30% +/- 8 in the outer 1-mm tumor shell only for groups 1, 2, 3, and 4, respectively. The ADC increase in group 4 was significantly higher (P = .0008 and P = .0189 for whole tumor volume and peripheral region, respectively) than that in group 1 on day 3, whereas tumor size did not significantly differ. At day 3, the dose-dependent ADC increases were linearly proportional to apoptotic cell and cleaved caspase-3 densities and were inversely proportional to the density of cells showing Ki67 expression. CONCLUSION Diffusion-weighted imaging enabled measurement of early breast tumor response to TRA-8 treatment, prior to detectable tumor shrinkage, providing an effective mechanism to noninvasively monitor TRA-8 efficacy. SUPPLEMENTAL MATERIAL http://radiology.rsnajnls.org/cgi/content/full/248/3/844/DC1.

The status of contemporary image-guided modalities in oncologic surgery.

OBJECTIVE To review the current trends in optical imaging to guide oncologic surgery. BACKGROUND Surgical resection remains the cornerstone of therapy for patients with early stage solid malignancies and more than half of all patients with cancer undergo surgery each year. The technical ability of the surgeon to obtain clear surgical margins at the initial resection remains crucial to improve overall survival and long-term morbidity. Current resection techniques are largely based on subjective and subtle changes associated with tissue distortion by invasive cancer. As a result, positive surgical margins occur in a significant portion of tumor resections, which is directly correlated with a poor outcome. METHODS A comprehensive review of studies evaluating optical imaging techniques is performed. RESULTS A variety of cancer imaging techniques have been adapted or developed for intraoperative surgical guidance that have been shown to improve functional and oncologic outcomes in randomized clinical trials. There are also a large number of novel, cancer-specific contrast agents that are in early stage clinical trials and preclinical development that demonstrate significant promise to improve real-time detection of subclinical cancer in the operative setting. CONCLUSIONS There has been an explosion of intraoperative imaging techniques that will become more widespread in the next decade.

Beyond the margins: real-time detection of cancer using targeted fluorophores

Over the past two decades, synergistic innovations in imaging technology have resulted in a revolution in which a range of biomedical applications are now benefiting from fluorescence imaging. Specifically, advances in fluorophore chemistry and imaging hardware, and the identification of targetable biomarkers have now positioned intraoperative fluorescence as a highly specific real-time detection modality for surgeons in oncology. In particular, the deeper tissue penetration and limited autofluorescence of near-infrared (NIR) fluorescence imaging improves the translational potential of this modality over visible-light fluorescence imaging. Rapid developments in fluorophores with improved characteristics, detection instrumentation, and targeting strategies led to the clinical testing in the early 2010s of the first targeted NIR fluorophores for intraoperative cancer detection. The foundations for the advances that underline this technology continue to be nurtured by the multidisciplinary collaboration of chemists, biologists, engineers, and clinicians. In this Review, we highlight the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, and consider the unique challenges associated with their effective application in clinical settings.

Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report

Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both Food and Drug Administration approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and the safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (www.isigs.org) on February 6, 2015, in Miami, Florida, and reflects a consensus of the participants’ opinions. Our objective was to critically evaluate the imaging platform technology and optical imaging agents and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.

Microbubble-mediated ultrasonic techniques for improved chemotherapeutic delivery in cancer

Background: Ultrasound (US) exposed microbubble (MB) contrast agents have the capability to transiently enhance cell membrane permeability. Using this technique in cancer treatment to increase the efficiency of chemotherapy through passive, localized delivery has been an emerging area of research. Purpose: Investigation of the influence of US parameters on MB-mediated drug delivery in cancer. Methods: The 2LMP breast cancer cells were used for in vitro experiments and 2LMP tumor-bearing mice were used during in vivo experiments. Changes in membrane permeability were investigated after the influence of MB-mediated US therapy parameters (i.e. frequency, mechanical index, pulse repetition period, US duration, and MB dosing and characteristics) on cancer cells. Calcein, a non-permeable fluorescent molecule, and Taxol, chemotherapeutic, were used to evaluate membrane permeability. Tumor response was also assessed histologically. Results: Combination chemotherapy and MB-mediated US therapy with optimized parameters increased cancer cell death by 50% over chemotherapy alone. Discussion: Increased cellular uptake of chemotherapeutic was dependent upon US system parameters. Conclusion: Optimized MB-mediated US therapy has the potential to improve cancer patient response to therapy via increased localized drug uptake, which may lead to a lowering of chemotherapeutic drug dosages and systemic toxicity.

Early Therapy Evaluation of Combined Anti–Death Receptor 5 Antibody and Gemcitabine in Orthotopic Pancreatic Tumor Xenografts by Diffusion-Weighted Magnetic Resonance Imaging

Early therapeutic efficacy of anti-death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5-7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4-5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 mug), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 +/- 9% (mean +/- SE) and 27 +/- 3%, respectively, significantly higher (P < 0.05) than those of group 1 (-1 +/- 5%) and group 2 (-2 +/- 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 +/- 0.4%, 0.6 +/- 0.2%, 3.1 +/- 0.9%, and 4.7 +/- 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy.

Determination of Breast Cancer Response to Bevacizumab Therapy Using Contrast-Enhanced Ultrasound and Artificial Neural Networks

Objective. The purpose of this study was to evaluate contrast‐enhanced ultrasound and neural network data classification for determining the breast cancer response to bevacizumab therapy in a murine model. Methods. An ultrasound scanner operating in the harmonic mode was used to measure ultrasound contrast agent (UCA) time‐intensity curves in vivo. Twenty‐five nude athymic mice with orthotopic breast cancers received a 30‐μL tail vein bolus of a perflutren microsphere UCA, and baseline tumor imaging was performed using microbubble destruction‐replenishment techniques. Subsequently, 15 animals received a 0.2‐mg injection of bevacizumab, whereas 10 control animals received an equivalent dose of saline. Animals were reimaged on days 1, 2, 3, and 6 before euthanasia. Histologic assessment of excised tumor sections was performed. Time‐intensity curve analysis for a given region of interest was conducted using customized software. Tumor perfusion metrics on days 1, 2, 3, and 6 were modeled using neural network data classification schemes (60% learning and 40% testing) to predict the breast cancer response to therapy. Results. The breast cancer response to a single dose of bevacizumab in a murine model was immediate and transient. Permutations of input to the neural network data classification scheme revealed that tumor perfusion data within 3 days of bevacizumab dosing was sufficient to minimize the prediction error to 10%, whereas measurements of physical tumor size alone did not appear adequate to assess the therapeutic response. Conclusions. Contrast‐enhanced ultrasound may be a useful tool for determining the response to bevacizumab therapy and monitoring the subsequent restoration of blood flow to breast cancer.

Endotoxin-induced proteolytic reduction in hepatic growth hormone (GH) receptor: a novel mechanism for GH insensitivity.

GH is an important anabolic hormone. We previously demonstrated in cell culture that the cell surface GH receptor (GHR) is susceptible to inducible metalloproteolytic cleavage that yields the shed receptor extracellular domain (called GH binding protein) and renders the cells desensitized to subsequent GH stimulation. Sepsis and inflammatory states are associated with hepatic desensitization to GH, although disparate mechanisms have been postulated in various animal models. Using C3H/HeJ mice, we now demonstrate that administration of lipopolysaccharide (LPS) causes marked hepatic desensitization to GH, assessed by monitoring signal transducer and activator of transcription 5 tyrosine phosphorylation and nuclear accumulation and with a novel noninvasive bioluminescence imaging system to track in vivo hepatic GH signaling serially in individual mice. This endotoxin-induced desensitization was accompanied by marked loss of hepatic GHR, which was not explained by changes in GHR mRNA abundance. Furthermore, we observe that LPS causes GH-binding protein shedding of a hepatically expressed wild-type GHR but not a GHR with a mutation in the metalloprotease cleavage site. These data suggest that in this model system, LPS-induced desensitization to GH is associated with proteolytic GHR cleavage. These data are the first to demonstrate inducible in vivo GHR proteolysis and suggest this is a mechanism to regulate GH sensitivity and its anabolic effects during sepsis or inflammation.