Curative surgery for other many cancers requires that the tumor be removed with a zone of normal tissue surrounding the tumor with ‘negative’ margins. Sarcomas, cancers of the bones, muscles, and fat, require WLE for cure. Unfortunately, ‘positive’ margins occur in 20-25% of sarcoma surgeries, associated with cancer recurrence and reduced survival. Our group successfully tested a small-molecule fluorophore (ABY-029) in sarcomas that targets the epidermal growth factor receptor. We sought to evaluate human sarcoma xenografts for epidermal growth factor receptor expression and binding of ABY-029 with and without exposure to standard presurgical chemotherapy and radiation. We inoculated groups of 24 NSG mice with five cell lines (120 mice total). Eight mice from each cell line received: 1) radiation alone; 2) chemotherapy alone; or 3) chemotherapy and radiation. We administered ABY-029 2-4 hours before surgery. Tumor and biopsy portions of background tissues were removed. All tissues were imaged on a LI-COR Odyssey and processed in pathology. There were no significant reductions in epidermal growth factor receptor expression or in ABY-029-mediated fluorescence in tumors exposed to chemotherapy, radiation, or both. fluorescence-guided surgery demonstrates strong promise to improve curative surgical cancer care, particularly for sarcomas where the positive margin rate is substantial. Fluorophore performance must be evaluated under circumstances that duplicate accurately the biological milieu relevant to a particular cancer. This work shows that human sarcoma xenografts subjected to standard therapies do not demonstrate a change in epidermal growth factor receptor expression or in epidermal growth factor receptor-targeted fluorescence, thereby indicating that epidermal growth factor receptor-targeted fluorescence-guided surgery should be feasible under normal therapeutic conditions in the clinic.
The status of lymph node is considered a critical prognostic factor for staging and guiding the future adjuvant treatment in many cancer types. The estimation of undetected micrometastases (0.2-2mm diameter) by conventional pathology was around 30-60% cases which has created a demand for the development of more fast and accurate approaches. In response, a paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic cancer cells in lymph nodes to guide pathology sectioning. To identify the most feasible and effective protocol using this approach to detect micrometastases intraoperatively, swine cervical lymph nodes were used to evaluate the potential of different protocols for the agents to diffuse into and out of intact nodes. Aby-029, an anti-EGFR affibody molecule labeled with IRDye-800CW was used as targeted imaging agent, and the IRDye-700DX carboxylate was used as control agent. The time-course paired-agent fluorescence of whole lymph node were recorded to monitor the uptake and washout kinetics. Subsequently, lymph nodes were frozen-sectioned and imaged under an 85-um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. After much trial-and error, the intranodal infusion staining and rinsing protocol demonstrated promising results that both imaging agents shown strong correlation with each other in the absence of cancer cells (r=0.99, p<0.001). This methodology indicated the potential of using paired-agent imaging approach to allow rapid and sufficient detection of micrometastases in excised lymph nodes intraoperatively.
Osteosarcoma is the most common primary malignant bone tumor in children. Patient survival with osteosarcoma is heavily influenced by the response to chemotherapy, measured by tumor necrosis upon histological analysis. Unfortunately, response is not measurable until the time of surgery and therefore modifications to chemotherapy protocol are only made after several weeks of treatment and surgery. Osteosarcoma tumors often demonstrate increased mineralization following the onset of chemotherapy. Furthermore, it has been hypothesized that this mineralization—apparent on radiographs—may correlate with chemotherapy response, however, this has not been demonstrated with qualitative visual evaluation. The ability to non-invasively measure a patient’s response to chemotherapy using plain radiographs, which is currently included in the normal clinical workflow, would guide the medical oncologists to tailor treatment for patients with osteosarcoma. After obtaining appropriate multi-center institutional review board approvals, we identified 31 patients that possess a pair of pre- and post-chemotherapy radiograph along with the necrosis measure. The images were digitized scans of physical radiographs between 1999 and 2013. Software was designed to measure the signal intensities in the tumor, a region of the soft tissue, air, and healthy bone. The tumor signals were normalized based on the random combination of air, soft tissue or bone, by subtraction or division. The differences in tumor signal between pre- and post-image were plotted against the percent necrosis determined by histological analysis. Different combinations of the normalization methods were compared based on the slope, coefficient of determination (R2) and Pearson correlation coefficient (ρ).
Surgical excision via wide local excision (WLE) of the primary sarcoma tumor is a mainstay of treatment due to the limited effectiveness of chemotherapy and radiation. Even with attempts at WLE, 22-34% of the patient will be diagnosed with a positive margin by the pathologist, necessitating additional radiation or surgery. Recent studies have demonstrated reduced local recurrence when using fluorescence-guided surgery (FGS) to detect residual sarcoma following attempted WLE.
ABY-029 is an anti-EGFR Affibody molecule labeled with IRDye800CW that is currently under Phase 0 human trial for FGS. To date, several studies have been performed to evaluate ABY-029 signal intensity in untreated human sarcoma xenografts; however, many patients undergoing cancer surgery have received pre-operative radiation and/or chemotherapy, which can affect tissue properties and tumor molecule expression level. Determining the effects of radiation and chemotherapy exposure on fluorophore binding in sarcomas may influence best practices in implementing FGS for sarcoma.
In this project, fluorophore signal intensities in tumor and surrounding tissue were measured and compared to the receptor concentration determined by immunohistochemistry. Here, we report the result for one EGFR positive synovial sarcoma cell lines, SW982. Four groups of human dose equivalent therapies – control, radiation, chemotherapy (Doxorubicin) and radiation followed by chemotherapy – were given to the tumor-bearing mice. The difference between groups can be used to determine the effects of preoperative sarcoma therapies on EGFR expression, ABY-029 uptake, and optical properties of tissues.
Fluorescent molecular-guided surgery (FGS) is at a tipping point in terms of clinical approval and adoption in a number cancer applications, with ongoing phase 0 and phase 1 clinical trials being carried out in a wide range of cancers using a wide range of agents. The pharmacokinetics of each of these agents and the physiology of these cancers can differ vastly on a patient-to-patient basis, bringing to question: how can one fairly compare different methodologies (defined as the combination of imaging agent, system, and protocol) and how can existing methodologies be further optimized? To this point, little methodology comparison has been carried out, and the majority of FGS optimization has concerned system development—on the level of maximizing signal-to-noise, dynamic detection range, and sensitivity—independently from traditional agent development—in terms of fluorophore brightness, toxicity, solubility, and binding affinity and specificity. Here we propose an inclusion of tumor and healthy tissue physiology (blood flow, vascular permeability, specific and nonspecific binding sites, extracellular matrix, interstitial pressure, etc.) variability into the optimization process and re-establish well-described task-based metrics for methodology optimization and comparing quality of one methodology to another. Two salient conclusions were identified: (1) contrast-tobackground variability is a simple metric that correlates with difficult-to-carry-out task-based metrics for comparing methodologies, and (2) paired-agent imaging protocols offer unique advantages over singleimaging- agent studies for mitigating confounding tumor and background physiology variability.
Long-term survival of head and neck squamous cell carcinoma (HNSCC) patients have proven to be correlated with negative surgical margins. Paired-Agent Imaging for Resection during Surgery (PAIRS) is capable of drawing the fine line between tumor and normal tissue by employing a control imaging-agent, which is co-administered with the targeted imaging agent to account for nonspecific signal. PAI is highly dependent on the parallel paired-agent delivery and static quantum yield of the agent to trace the molecular concentration. However, it is well known that nonspecific binding of fluorescence probes to plasma proteins can change its delivery, dissociation constant, and quantum yield.
A thorough evaluation of the effect of plasma protein binding in the estimation of receptor concentration was performed for the paired-agents in this study. We are planning to evaluate ABY-029, an anti-epithelial growth factor receptor (EGFR) Affibody, and IRDye 700DX as a control agent. The plasma-dependent change in fluorescence intensity, percent binding, and in vivo distribution kinetics will be studied for each agent alone, and in combination. In this proceeding, the absorption, emission patterns for the targeted agent, ABY-029, measured by UV-Vis, fluorometer, and Pearl were shown. Initial studies indicate that binding to Bovine serum albumin (BSA), human serum albumin (HSA) and EGFR can introduce the Solvatochromic shift, which will change the absorption and emission pattern for ABY-029. Computational modeling will be performed to determine how each of these changes will affect the determined BP, and thus detection of tumors from normal tissue.
Sentinel lymph node involvement is recognized as a prognostic factor in breast cancer staging and is essential to guide optimal treatment. The possibility of missed micrometastases by using conventional methods was estimated around 20-60% cases has created a demand for the development of more accurate approaches. A paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, lymph node micrometastases biological tissue model was developed and were stained with targeted and control imaging agent solution to evaluate the binding potential of the agents of intact nodes. ABY-029, an EGFR specific affibody was labeled with IRDye-800CW(LICOR) as targeted agent and IRDye-700DX was hydrolyzed as control agent. Lymph nodes phantoms were stained for 60 min, followed by 60 min rinsing, and the fluorescence of whole lymph node phantoms were recorded to evaluate the spatial distribution of both agents in the entire phantom. Measured binding potential of targeted agent between micrometastases and control regions were 0.652 ± 0.130 and -0.008 ± 0.042 respectively (p < 0.0001). The results demonstrate the potential to enhance the sensitivity of lymph node pathology using paired-agent imaging in a whole human lymph node.
The presence of lymph node metastases played as a critical prognostic factor in breast cancer treatment and guiding the future adjuvant treatment. The possibility of missed micrometastases by conventional pathology was estimated around 20-60% cases has created a demand for the development of more accurate approaches. Here, a paired-agent imaging approach is presented that employs a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, healthy rat and human lymph nodes were stained with targeted and control imaging agent solution to evaluate the potential for the agents to diffuse into and out of intact nodes. Erbitux, an EGFR specific antibody was labeled with IRDye-700DX(LICOR) as targeted agent and IRDye-800CW was labeled to rat IgG as control agent. Lymph nodes were stained for 60 min, followed by 30 min rinsing, and the uptake and washout of fluorescence were recorded. Subsequently, lymph nodes were frozen-sectioned and imaged under an 80- um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. Both imaging agents correlated well with each other(r=0.877) and the binding potential of targeted agent was found to be 0.08 ± 0.22 along the lymph node in the absence of binding. The results demonstrate this approach’s potential to enhance the sensitivity of lymph node pathology by detecting fewer than 1000 cell in a whole human lymph node.
Intraoperative tumor/surgical margin assessment is required to achieve higher tumor resection rate in breast-conserving surgery. Though current histology provides incomparable accuracy in margin assessment, thin tissue sectioning and the limited field of view of microscopy makes histology too time-consuming for intraoperative applications. If thick tissue, wide-field imaging can provide an acceptable assessment of tumor cells at the surface of resected tissues, an intraoperative protocol can be developed to guide the surgery and provide immediate feedback for surgeons. Topical staining of margins with cancer-targeted molecular imaging agents has the potential to provide the sensitivity needed to see microscopic cancer on a wide-field image; however, diffusion and nonspecific retention of imaging agents in thick tissue can significantly diminish tumor contrast with conventional methods. Here, we present a mathematical model to accurately simulate nonspecific retention, binding, and diffusion of imaging agents in thick tissue topical staining to guide and optimize future thick tissue staining and imaging protocol. In order to verify the accuracy and applicability of the model, diffusion profiles of cancer targeted and untargeted (control) nanoparticles at different staining times in A431 tumor xenografts were acquired for model comparison and tuning. The initial findings suggest the existence of nonspecific retention in the tissue, especially at the tissue surface. The simulator can be used to compare the effect of nonspecific retention, receptor binding and diffusion under various conditions (tissue type, imaging agent) and provides optimal staining and imaging protocols for targeted and control imaging agent.
Sentinel lymph node status is a critical prognostic factor in breast cancer treatment and is essential to guide future adjuvant treatment. The estimation that 20-60% of micrometastases are missed by conventional pathology has created a demand for the development of more accurate approaches. Here, a paired-agent imaging approach is presented that employs a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, healthy pig lymph nodes were stained with targeted and control imaging agent solution to evaluate the potential for the agents to diffuse into and out of intact nodes. Aby-029, an anti-EGFR affibody was labeled with IRDye 800CW (LICOR) as targeted agent and IRDye 700DX was hydrolyzed as a control agent. Lymph nodes were stained and rinsed by directly injecting the agents into the lymph nodes after immobilization in agarose gel. Subsequently, lymph nodes were frozen-sectioned and imaged under an 80-um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. The binding potentials were acquired by a pixel-by-pixel calculation and was found to be 0.02 ± 0.06 along the lymph node in the absence of binding. The results demonstrate this approach’s potential to enhance the sensitivity of lymph node pathology by detecting fewer than 1000 cell in a whole human lymph node.
Clinical symptoms of diabetic retinopathy are not detectable until damage to the retina reaches an irreversible stage, at least by today’s treatment standards. As a result, there is a push to develop new, “sub-clinical” methods of predicting the onset of diabetic retinopathy before the onset of irreversible damage. With diabetic retinopathy being associated with the accumulation of long-term mild damage to the retinal vasculature, retinal blood vessel permeability has been proposed as a key parameter for detecting preclinical stages of retinopathy. In this study, a kinetic modeling approach used to quantify vascular permeability in dynamic contrast-enhanced medical imaging was evaluated in noise simulations and then applied to retinal videoangiography data in a diabetic rat for the first time to determine the potential for this approach to be employed clinically as an early indicator of diabetic retinopathy. Experimental levels of noise were found to introduce errors of less than 15% in estimates of blood flow and extraction fraction (a marker of vascular permeability), and fitting of rat retinal fluorescein angiography data provided stable maps of both parameters.
Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional “single-tracer” molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.
Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, “untargeted” imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.
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