Safety of panitumumab-IRDye800CW and cetuximab-IRDye800CW for fluorescence-guided surgical navigation in head and neck cancers

Purpose: To demonstrate the safety and feasibility of leveraging therapeutic antibodies for surgical imaging. Procedures: We conducted two phase I trials for anti-epidermal growth factor receptor antibodies cetuximab-IRDye800CW (n=12) and panitumumab-IRDye800CW (n=15). Adults with biopsy-confirmed head and neck squamous cell carcinoma scheduled for standard-of-care surgery were eligible. For cetuximab-IRDye800CW, cohort 1 was intravenously infused with 2.5 mg/m(2), cohort 2 received 25 mg/m(2), and cohort 3 received 62.5 mg/m(2). For panitumumab-IRDye800CW, cohorts received 0.06 mg/kg, 0.5 mg/kg, and 1 mg/kg, respectively. Electrocardiograms and blood samples were obtained, and patients were followed for 30 days post-study drug infusion. Results: Both fluorescently labeled antibodies had similar pharmacodynamic properties and minimal toxicities. Two infusion reactions occurred with cetuximab and none with panitumumab. There were no grade 2 or higher toxicities attributable to cetuximab-IRDye800CW or panitumumab-IRDye800CW; fifteen grade 1 adverse events occurred with cetuximab-I RDye800CW, and one grade 1 occurred with panitumumab-IRDye800CW. There were no significant differences in QTc prolongation between the two trials (p=0.8). Conclusions: Panitumumab-IRDye800CW and cetuximab-IRDye800CW have toxicity and pharmacodynamic profiles that match the parent compound, suggesting that other therapeutic antibodies may be repurposed as imaging agents with limited preclinical toxicology data

Fluorescence imaging to localize head and neck squamous cell carcinoma for enhanced pathological assessment

Accurately identifying close or positive margins in real-time permits re-excision during surgical procedures. Intraoperative assessment of margins via gross examination and frozen section is a widely used tool to assist the surgeon in achieving complete resection. While this methodology permits diagnosis of freshly resected tissue, the process is fraught with misinterpretation and sampling errors. During fluorescence-guided surgery, an exogenous fluorescent agent specific for the target disease is imaged in order to navigate the surgical excision. As this technique quickly advances into the clinic, we hypothesize that the disease-specific fluorescence inherently contained within the resected tissues can be used to guide histopathological assessment. To evaluate the feasibility of fluorescence-guided pathology, we evaluated head and neck squamous cell carcinoma tumour specimens and margins resected from animals and patients after systemic injection of cetuximab-IRDye800CW. In a preclinical model of luciferase-positive tumour resection using bioluminescence as the gold standard, fluorescence assessment determined by closed-field fluorescence imaging of fresh resected margins accurately predicted the presence of disease in 33/39 positive margins yielding an overall sensitivity of 85%, specificity of 95%, positive predictive value (PPV) of 94%, and a negative predictive value (NPV) of 87%, which was superior to both surgical assessment (54%, 61%, 57%, and 58%) and pathological assessment (49%, 95%, 91%, and 66%), respectively. When the power of the technique was evaluated using human-derived tumour tissues, as little as 0.5mg (1mm(3)) of tumour tissue was identified (tumour-to-background-ratio:5.2). When the sensitivity/specificity of fluorescence-guided pathology was determined using traditional histological assessment as the gold standard in human tissues obtained during fluorescence-guided surgery, the technique was highly accurate with a sensitivity of 91%, specificity of 85%, PPV of 81%, and NPV of 93% for 90 human-derived samples. This approach can be used as a companion to the pathologist, eliminating confounding factors while impacting surgical intervention and patient management

In Vivo Fluorescence Immunohistochemistry:Localization of Fluorescently Labeled Cetuximab in Squamous Cell Carcinomas

Anti-EGFR (epidermal growth factor receptor) antibody based treatment strategies have been successfully implemented in head and neck squamous cell carcinoma (HNSCC). Unfortunately, predicting an accurate and reliable therapeutic response remains a challenge on a per-patient basis. Although significant efforts have been invested in understanding EGFR-mediated changes in cell signaling related to treatment efficacy, the delivery and histological localization in (peri-) tumoral compartments of antibody-based therapeutics in human tumors is poorly understood nor ever made visible. In this first in-human study of a systemically administered near-infrared (NIR) fluorescently labeled therapeutic antibody, cetuximab-IRDye800CW (2.5 mg/m(2), 25 mg/m(2), and 62.5 mg/m(2)), we show that by optical molecular imaging (i.e. denominated as In vivo Fluorescence Immunohistochemistry) we were able to evaluate localization of fluorescently labeled cetuximab. Clearly, optical molecular imaging with fluorescently labeled antibodies correlating morphological (peri-) tumoral characteristics to levels of antibody delivery, may improve treatment paradigms based on understanding true tumoral antibody delivery

Breast Cancer Imaging Using the Near-Infrared Fluorescent Agent, CLR1502

Positive margins after breast conservation surgery represent a significant problem in the treatment of breast cancer. The near-infrared fluorescence agent CLR1502 (Cellectar Biosciences, Madison, WI) was studied in a preclinical breast cancer model to determine imaging properties and ability to detect small islands of malignancy. Nude mice bearing human breast cancer flank xenografts were given a systemic injection of CLR1502, and imaging was performed using LUNA (Novadaq Technologies Inc., Richmond, BC) and Pearl Impulse (LI-COR Biosciences, Lincoln, NE) devices. Normal tissues were examined for fluorescence signal, and conventional and fluorescence histology was performed using the Odyssey scanner. Peak tumor to background ratio occurred 2 days after injection with CLR1502. The smallest amount of tumor that was imaged and detected using these devices was 1.9 mg, equivalent to 1.9 × 106 cells. The highest fluorescence signal was seen in tumor and normal lymph node tissue, and the lowest fluorescence signal was seen in muscle and plasma. Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using CLR1502. This pilot study supports further investigations of this fluorescent agent for improving surgical resection of malignancies, with the goal of eventual clinical translation

Genetic strategy to decrease complement activation with adenoviral therapies.

BackgroundA major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection.MethodsA recombinant Ad5 vector was genetically modified to display a peptide sequence ("rH17d'"), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d' and Ad5.HVR5-rH17d' were constructed by engineering the rH17d' peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively.ResultsIn vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d', whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d' or Ad5.HVR5-rH17d' showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d' and His6 vectors following their intratumoral injection was similar.ConclusionsDisplaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism

Fibroblast Growth Factor Receptor Mediates Fibroblast-Dependent Growth in EMMPRIN-Depleted Head and Neck Cancer Tumor Cells

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth. Mol Cancer Res; 9(8); 1008-17. (C)2011 AACR

Fluorescence-guided resection of experimental malignant glioma using cetuximab-IRDye 800CW

The standard treatment for Glioblastoma Multiforme (GBM) remains maximal safe surgical resection. Here, we evaluated the ability of a systemically administered antibody-dye probe conjugate (cetuximab-IRDye 800CW) to provide sufficient fluorescent contrast for surgical resection of disease in both subcutaneous and orthotopic animal models of GBM. Multiple luciferase positive GBM cell lines (D-54MG, U-87MG, U-251MG; n=5) were implanted in mouse flank and tumours fluorescently imaged daily using a closed-field NIR system after cetuximab-IRDye 800CW systemic administration. Orthotopic models were also generated (n=5) and tumour resection was performed under white-light and fluorescence guidance using an FDA-approved wide-field NIR imaging system. Residual tumour was monitored using luciferase imaging. Immunohistochemistry was performed to characterize tumour fluorescence, epidermal growth factor receptor (EGFR) expression, and vessel density. Daily imaging of tumours revealed an average tumour-to-background (TBR) of 4.5 for U-87MG, 4.1 for D-54MG, and 3.7 for U-251MG. Fluorescence intensity within the tumours peaked on day-1 post cetuximab-IRDye 800CW administration, however the TBR increased over time in two of the three cell lines. For the orthotopic model, TBR on surgery day ranged from 19 to 23 during wide-field, intraoperative imaging. Surgical resection under white-light on day 3 post cetuximab-IRDye 800CW resulted in an average 41% reduction in luciferase signal while fluorescence-guided resection using wide-field NIR imaging resulted in a significantly (P=0.001) greater reduction in luciferase signal (87%). Reduction of luciferase signal was found to correlate (R(2)=0.99) with reduction in fluorescence intensity. Fluorescence intensity was found to correlate (P<0.05) with EGFR expression in D-54MG and U-251MG tumour types but not U-87MG. However, tumour fluorescence was found to correlate with vessel density for the U-87MG tumours. Here we show systemic administration of cetuximab-IRDye 800CW in combination with wide-field NIR imaging provided robust and specific fluorescence contrast for successful localization of disease in subcutaneous and orthotopic animal models of GBM

Hyperintensity of integrin-targeted fluorescence agent IntegriSense750 accurately predicts flap necrosis compared to Indocyanine green

BackgroundFlap necrosis is a feared complication of reconstructive surgery. Current methods of prediction using Indocyanine green (ICG) lack specificity. IntegriSense750 is a fluorescence agent that binds sites of vascular remodeling. We hypothesized that IntegriSense750 better predicts flap compromise compared to ICG.MethodsFifteen mice underwent lateral thoracic artery axial flap harvest. Mice received an injection of ICG (n = 7) or IntegriSense750 (n = 8) daily from postoperative days (POD) 0-3 and were imaged daily. Mean signal-to-background ratios quantified the change in fluorescence as necrosis progressed.ResultsMean signal-to-background ratio was significantly higher for IntegriSense750 compared to ICG on POD0 (1.47 ± 0.17 vs. 0.86 ± 0.21, p = 0.01) and daily through POD3 (2.12 ± 0.70 vs. 0.96 ± 0.29, p ConclusionsIntegriSense750 demonstrates increased signal-to-background ratio at areas of flap distress compared to ICG which may increase identification of flap necrosis and improve patient outcomes