Background Fluorescence imaging hardware (SPY) has recently been developed for intraoperative assessment of blood flow via detection of probes emitting in the near-infrared (NIR) spectrum. This study sought to determine if this imaging system was capable of detecting micrometastatic head and neck squamous cell carcinoma (HNSCC) in preclinical models. Methods A NIR fluorescent probe (IRDye800CW) was covalently linked to a monoclonal antibody targeting EGFR (panitumumab) or non-specific IgG. HNSCC flank (SCC-1) and orthotopic (FADU and OSC19) xenografts were imaged 48-96hrs following systemic injection of labeled panitumumab or IgG. The primary tumor and regional lymph nodes were dissected using fluorescence guidance with the SPY system and grossly assessed with a charge-coupled NIR system (Pearl). Histologic slides were also imaged with a NIR charged-coupled device (Odyssey) and fluorescence intensity was correlated with pathologic confirmation of disease. Results Orthotopic tongue tumors were clearly delineated from normal tissue with tumor-to-background ratios of 2.9(Pearl) and 2.3(SPY). Disease detection was significantly improved with panitumumab-IRDye compared to IgG-IRDye800 (P<0.05). Tissue biopsies (average size=3.7mm) positive for fluorescence were confirmed for pathologic disease by histology and immunohistochemistry (n=25/25). Biopsies of non-fluorescent tissue were proven to be negative for malignancy (n=28/28). The SPY was able to detect regional lymph node metastasis (<1.0mm) and microscopic areas of disease. Standard histological assessment in both frozen and paraffin-embedded histologic specimens was augmented using the Odyssey. Conclusions Panitumumab-IRDye800 may have clinical utility in detection and removal of microscopic HNSCC using existing intraoperative optical imaging hardware and may augment analysis of frozen and permanent pathology.

Objective To determine if microbubble-mediated ultrasound therapy (MB-UST) can improve cisplatin or cetuximab cytotoxicity of head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo by increasing tumor-specific drug delivery by disruption of tumor cell membranes and enhancing vascular permeability. Study Design In vitro and in vivo study. Setting University medical center. Subjects Immunodeficient mice (6 weeks old) and 4 HNSCC cell lines. Methods Changes to cell permeability were assessed in vitro after MB-UST. Cellular apoptosis resulting from adjuvant MB-UST with subtherapeutic doses of cisplatin or cetuximab was assessed by cell survival assays in vitro. The in vivo effect of adjuvant MB-UST in flank tumors was assessed in vivo with histological analysis and diffusion-weighted magnetic resonance imaging (DW-MRI). Results In vitro results revealed that MB-UST can increase cell permeability and enhance drug uptake and apoptosis in 4 HNSCC cell lines. In vivo adjuvant MB-UST with cetuximab or cisplatin showed a statistically significant reduction in tumor size when compared with untreated controls. TUNEL analysis yielded a larger number of cells undergoing apoptosis in tumors treated with cetuximab and adjuvant MB-UST than did cetuximab alone but was not significantly greater in tumors treated with cisplatin and adjuvant MBUST compared with cisplatin alone. DW-MRI analysis showed more free water, which corresponds to increased cell membrane disruption, in tumors treated with MB-UST. Conclusion MB-UST promotes disruption of cell membranes in tumor cells in vitro, which may be leveraged to selectively improve the uptake of conventional and targeted therapeutics in vivo.

Objective To assess the feasibility of panitumumab in real-time fluorescent imaging and histologic processing of cutaneous squamous cell carcinoma (cSCC) in mice. Design A near-infrared (NIR) fluorescent probe (IRDye800-CW) was covalently linked to a monoclonal antibody–targeting epidermal growth factor receptor (panitumumab) or nonspecific IgG and injected into mice bearing flank xenografts from a cSCC cell line (SCC-13 or SRB-12; n = 7), human split-thickness skin grafts (STSGs; n = 3), or a human tumor explant (n = 1). The tumor and lymph nodes were imaged and dissected using fluorescence guidance with the SPY imaging system and verified with a charge-coupled NIR system. An NIR scanning device (Odyssey) was used to measure fluorescence intensity in histological sections. Subjects Immunodeficient mice. Setting In vivo and in vitro imaging lab. Results Tumor tissue could be delineated from the human STSG with tumor-to-background ratios of 4.5 (Pearl) and 3.4 (SPY). Tumor detection was substantially improved with panitumumab-IRDye800 compared with IgG-IRDye800. Biopsies positive for fluorescence were assessed by histology and immunohistochemistry (n = 18/18) to confirm the presence of tumor, yielding a 100% sensitivity. Biopsies of non-fluorescent tissue negative for malignancy (n = 18/18) yielded a specificity of 100%. Furthermore, the SPY system was able to detect residual disease as small as 200 μm in diameter. In addition, the Odyssey confirmed fluorescence of microscopic disease (in tumor samples of frozen and paraffin-embedded histologic specimens) but not in adjacent noncancerous tissue. Conclusions These data suggest panitumumab-IRDye800 may have clinical utility in detection and removal of subclinical cSCC using Food and Drug Administration–approved imaging hardware.

Purpose Recurrent or advanced stage cutaneous squamous cell carcinoma (cSCC) is difficult to treat and available therapies have a high failure rate. Anti-epidermal growth factor receptor therapy (EGFR) is associated with a facial rash that may potentiate radiation related cutaneous toxicities. The objective of this clinical trial was to assess the toxicity profile of erlotinib therapy combined with postoperative adjuvant radiotherapy in patients with advanced cSCC. Methods This study is a single-arm, prospective phase I open-label study of erlotinib with radiation therapy (XRT) to treat 15 patients with advanced cutaneous head and neck squamous cell carcinoma. Toxicity data were summarized and survival was analyzed with the Kaplan-Meier method. Results The majority of patients were male (87%) and presented with T4 disease (93%). The most common toxicity attributed to erlotinib was a grade 2–3 dermatologic reaction occurring in 100% of the patients, followed by mucositis (87%). Diarrhea occurred in 20% of the patients. The two year recurrence rate was 26.7% and mean time to cancer recurrence was 10.5 months. Two year overall survival was 65% and disease free survival was 60%. Conclusions Erlotinib and radiation therapy had an acceptable toxicity profile in patients with advanced cutaneous squamous cell carcinoma. The disease-free survival in this cohort was comparable to historical controls.

Objective To investigate the feasibility of ultrasonographic (US) imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. We hypothesize that conjugation of microbubble contrast agent to tumor-specific antibodies may improve US detection of head and neck squamous cell carcinoma (HNSCC). Design Preclinical blinded assessment of anti-EGFR and anti-CD147 microbubble contrast agents for US imaging of HNSCC. Setting Animal study. Subjects Immunodeficient mice. Intervention Injection of targeted microbubbles. Main Outcome Measure Microbubble uptake in tumors as detected by US. Results In vitro assessment of anti–epidermal growth factor receptor (EGFR) and anti-CD147–targeted micro-bubbles in 6 head and neck cancer cell lines yielded a 6-fold improvement over normal dermal fibroblasts (P<.001). Binding of targeted agents had a positive correlation to both epidermal growth factor receptor (EGFR) (R2=0.81) and CD147 (R2=0.72) expression among all cell lines. In vivo imaging of flank tumors in nude mice (N=8) yielded enhanced resolution of anti-EGFR– and anti-CD147–targeted microbubble agents over IgG control (P<.001), while dual-targeted contrast agents offered enhanced imaging over single-targeted contrast agents (P=.02 and P=.05, respectively). In a blinded in vivo assessment, targeted contrast agents increased intratumoral enhancement of flank tumors over controls. Targeted US contrast agents to both EGFR and CD147 were 100% sensitive and 87% specific in the detection of flank tumors. Conclusion This preclinical study demonstrates feasibility of using molecular US to target HNSCC for contrast-enhanced imaging of HNSCC tumor in vivo.