No abstract

X-ray based computed tomography (CT), is among the most convenient imaging/diagnostic tools in hospitals today in terms of availability, efficiency and cost. However, in contrast to magnetic resonance imaging (MRI) and various nuclear medicine imaging modalities, CT is not considered a molecular imaging modality since targeted and molecularly specific contrast agents have not yet been developed. Here we describe a targeted molecular imaging platform that enables, for the first time, cancer detection at the cellular and molecular level with standard clinical CT. The method is based on gold nano-probes that selectively and sensitively target tumor selective antigens, while inducing distinct contrast in CT imaging (increased x-ray attenuation). We present an in vitro proof of principle demonstration for head and neck cancer, showing that the attenuation coefficient for the molecularly targeted cells is over 5 times higher than for identical but untargeted cancer cells or for normal cells. We expect this novel imaging tool to lead to significant improvements in cancer therapy, due to earlier detection, accurate staging and micro-tumor identification.Imaging plays a critical role in overall cancer management; in diagnostics, staging, radiation planning and evaluation of treatment efficiency. Standard clinical imaging modalities such as CT, MRI and ultrasound, can be categorized as structural imaging modalities; they are able to identify anatomical patterns and to provide basic information regarding tumor location, size and spread based on endogenous contrast. However, these imaging modalities are not efficient in detecting tumors and metastases that are smaller than 0.5 cm, and they can barely distinguish between benign and cancerous tumors 1 .Molecular imaging is an emerging field that integrates molecular biology with in vivo imaging, in order to gain information regarding biological processes and to identify diseases based on molecular markers, which usually appear before the clinical presentation of the disease. Currently, positron emission tomography and single photon emission tomography are the main molecular imaging modalities in clinical use, however, they provide only functional information regarding molecular processes and metabolites, which is indirect and nonspecific to distinct cells or diseases 2,3 . Recently, various types of targeted nano-probes have been developed for optical and MRI molecular imaging, such as superparamagnetic nanoparticles 4-7 ; quantum dots [8][9][10] and gold nanoparticles as cancer optical imaging probes [11][12][13] .

Application of immune cell-based therapy in routine clinical practice is challenging due to the poorly understood mechanisms underlying success or failure of treatment. Development of accurate and quantitative imaging techniques for noninvasive cell tracking can provide essential knowledge for elucidating these mechanisms. We designed a novel method for longitudinal and quantitative in vivo cell tracking, based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with state-of-the-art nanotechnology. Herein, T-cells were transduced to express a melanoma-specific T-cell receptor and then labeled with gold nanoparticles (GNPs) as a CT contrast agent. The GNP-labeled T-cells were injected intravenously to mice bearing human melanoma xenografts, and whole-body CT imaging allowed examination of the distribution, migration, and kinetics of T-cells. Using CT, we found that transduced T-cells accumulated at the tumor site, as opposed to nontransduced cells. Labeling with gold nanoparticles did not affect T-cell function, as demonstrated both in vitro, by cytokine release and proliferation assays, and in vivo, as tumor regression was observed. Moreover, to validate the accuracy and reliability of the proposed cell tracking technique, T-cells were labeled both with green fluorescent protein for fluorescence imaging, and with GNPs for CT imaging. A remarkable correlation in signal intensity at the tumor site was observed between the two imaging modalities, at all time points examined, providing evidence for the accuracy of our CT cell tracking abilities. This new method for cell tracking with CT offers a valuable tool for research, and more importantly for clinical applications, to study the fate of immune cells in cancer immunotherapy.

The dose-volume-outcome data for RT-associated laryngeal edema, laryngeal dysfunction, and dysphagia, have only recently been addressed, and are summarized. For late dysphagia, a major issue is accurate definition and uncertainty of the relevant anatomical structures. These and other issues are discussed. Radiation therapy (RT) is the primary modality allowing larynx preservation in patients with tumors in the upper aero-digestive tract. RT-induced laryngeal edema (due to inflammation and lymphatic disruption) is a common and expected side effect. Progressive edema and associated fibrosis can lead to long-term problems with phonation and swallowing (1). Since the primary goal of larynx preservation is speech/swallowing retention, RT-induced laryngeal dysfunction may undermine this therapeutic approach. In many instances the larynx and pharynx are target structures and purposefully receive high RT doses.Dysphagia is common after chemo-irradiation of Head-and-Neck (HN) cancer. For example, patients on RTOG 91-11 were randomized to receive RT +/− concurrent cisplatin. The combined modality arm demonstrated improved tumor control rates (2). However, one year after therapy, 23% of the patients in the chemo-RT arm were unable to eat solid food, vs. with 9% with RT alone. Aspiration pneumonia associated with dysphagia after intensive chemo-

Purpose To assess whether alterations in tumor blood volume (BV) and flow (BF) during the early course of chemo-radiation therapy (RT) for head and neck cancer (HNC) predict treatment outcome. Methods Fourteen patients receiving concomitant chemo-RT for non-resectable, locally advanced HNC underwent dynamic-contrast enhanced (DCE) MRI scans pre-therapy and two weeks after initiation of chemo-RT. BV and BF were quantified from DCE MRI. Pre-RT BV and BF as well as their changes during RT were evaluated separately in the primary gross tumor volume (GTV) and nodal GTV for association with outcomes. Results At a median follow-up of ten months (range of 5 – 27 months), nine patients had local-regional controlled disease (LRC). One patient had regional failure (RF), three had local failures (LF) and one had local-regional failure (LRF). Reduction in tumor volume after 2 weeks of chemo-RT did not predict for local control. In contrast, the BV in the primary GTV after 2 weeks of chemo-RT was increased significantly in the LC patients compared to the LF patients (p<0.03). Conclusions Our data suggest that an increase in available primary tumor blood for oxygen extraction during the early course of RT is associated with local control, thus yielding a predictor with potential to modify treatment. These findings require validation in larger studies.

Purpose-Dysfunction of pharyngeal constrictors (PCs) after chemo-irradiation of head and neck (HN) cancer has been proposed as major cause of dysphagia. We conducted prospective MRI study to evaluate anatomical changes in the PCs after chemoirradiation, to gain insight of the mechanism of their dysfunction and their dose-effect relationships. The PCs were compared to the sternocleidomastoid muscles (SCMs), which receive high doses but do not relate to swallowing.Patients and Methods-Twelve patients with stage III-IV HN cancer underwent MRI before and 3 months after the completing chemo-irradiation. T1-and T2-weighted signals and muscle thickness were evaluated for PCs (superior, middle, and inferior), and SCMs. Mean muscle doses were determined after registration with the planning CT.Results-T1-weighted signals decreased in both PCs and SCMs receiving >50 Gy (p<0.03), but not in muscles receiving lower doses. T2-weighted signals in the PCs increased significantly as the dose increased (R 2 =0.34, p=0.01). The T2 signal changes in the PCs were significantly higher than the T2 changes in the SCMs (p<0.001). Increased thickness was noted in all PCs, with muscles receiving >50 Gy gaining significantly more thickness than PCs receiving lesser doses (p=0.02). In contrast, the SCM thickness decreased post-therapy (p=0.002).Conclusions-These MRI-based findings, notably the differences between PCs and SCMs, suggest that underlying causes of PC dysfunction are inflammation and edema, likely consequential to acute mucositis affecting the submucosa -lying PCs. These results support reducing mean PC doses to ≤50 Gy, as well as reducing acute mucositis, to improve long-term dysphagia.

A major problem in the treatment of head and neck cancer today is the resistance of tumors to traditional radiation therapy, which results in 40% local failure, despite aggressive treatment. The main objective of this study was to develop a technique which will overcome tumor radioresistance by increasing the radiation absorbed in the tumor using cetuximab targeted gold nanoparticles (GNPs), in clinically relevant energies and radiation dosage. In addition, we have investigated the biological mechanisms underlying tumor shrinkage and the in vivo toxicity of GNP. The results showed that targeted GNP enhanced the radiation effect and had a significant impact on tumor growth (P < 0.001). The mechanism of radiation enhancement was found to be related to earlier and greater apoptosis (TUNEL assay), angiogenesis inhibition (by CD34 level) and diminished repair mechanism (PCNA staining). Additionally, GNPs have been proven to be safe as no evidence of toxicity has been observed.

Purpose Re-irradiation (re-RT) of recurrent head and neck cancer (HNC) may achieve long term disease control in some patients, at the expense of high rates of late sequelae. Limiting the re-RT targets to the recurrent gross tumor volume (rGTV) would reduce the volumes of re-irradiated tissues, however, its effect on tumor recurrence pattern is unknown. Methods Retrospective review of 66 patients who underwent curative-intent re-RT for non-resectable recurrent or second primary mucosal squamous cell HNC. Treatment was delivered with 3-dimensional conformal (3D) RT or intensity modulated RT (IMRT). The targets in all patients consisted of the rGTVs with tight (0.5 cm) margins, with no intent to treat prophylactically lymph nodes or sub-clinical disease in the vicinity of the rGTVs. The sites of local-regional failures (LRFs) were determined using imaging at the time of failure, and were compared to the rGTVs. Results Median re-RT dose was 68 Gy. 47 patients (71%) received concomitant chemotherapy and 31 (47%) received hyperfractionated, accelerated RT. At a median follow up 42 month, 16 (23%) are alive and free of disease. Fifty patients (77%) had a third recurrence or persistent disease, including 47 LRFs. All LRFs occurred within the rGTVs except for two (4%) (95% C.I. 0; 11 %). Nineteen patients (29%) had grade ≥3 late complications, mostly dysphagia (12 patients). Conclusion Almost all LRFs occurred within the re-irradiated rGTVs despite avoiding prophylactic RT of tissue at risk of subclinical disease. These results support confining the re-RT targets to the rGTVs to reduce re-irradiated tissue volumes.