This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The efficacy of radiofrequency ablation (RFA) in destroying tumor is well established. However, it has been mostly utilized in palliation, for unresectable disease, or lesions in non-surgical candidates. We believe in a broader indication for using RFA in the cancer treatment.Currently, a variety of cancer vaccines are being developed. Although it is now accepted that patients can mount an immune response to their cancer, approaches to enhance this effect have met with limited success and only anecdotal clinical results. New methods to activate the immune response more effectively are required. RFA, by using electrical energy to heat and destroy tumor, releases denatured proteins, peptides and heat shock proteins that can act as antigen sources for activating tumor antigen-specific T cell and antibody responses. This suggests that RFA can result in anti-tumor immune responses. This has, in fact, been demonstrated in animal models. We hypothesize that in addition to producing such an anti-tumor response, RFA, through the same mechanism, will potentiate the action of immunotherapy.Our experimental design is simple but powerful. We will implant C57BL/6 mice on both flanks with tumor cells from the mouse colon cancer line MCA-38-CEA-2-XY. Once the tumors reach approximately 1 cm in diameter (approximately 15 days), the mice will be randomized to four groups. The mice in Group A (control Group, n=10) will undergo a sham RFA procedure to one flank tumor, involving an incision being created and sealed but no RFA. This will be our control group. The mice in Group B (RFA only group, n=20) will undergo RFA on the tumor in one flank. This group will allow us to examine the anti-tumor effect of RFA alone, both locally and systemically through the effect on the treated tumor and the untreated tumor respectively. The mice in Group C (vaccine alone group, n = 20) will undergo the sham RFA procedure and also receive GM-CSF and IL-2 injection for 5 days. As the generation of anti-tumor immunity following the administration of GM-CSF and IL-2 has already been well documented, this group will establish the anti-tumor effect of vaccine alone in this specific experimental model. Finally, the mice in Group D (combination treatment group, n = 20) will undergo both RFA on the tumor in one flank as well as receive GM-CSF and IL-2 injections for 5 days. This group will test our hypothesis that the combined anti-tumor effect of RFA and vaccine will be complementary in their actions. We will evaluate the effects of treatment through tumor size, MR perfusion and imaging characteristics, and immunoassays. For the immune assays, we will look at anti-tumor antibody responses using a flow cytometric assay and T cell responses against the tumor by ELISPOT. Due to the possibility that a clear anti-tumor immune response may not be detected by these methods, tumor growth and perfusion inhibition will be key endpoints to establish synergy between immune stimulus and RFA. Tumor size will be measured by Vernier calipers every two days. This is an accepted method in assessing for interval change in tumors experimentally treated with vaccine and thus needs to be included in our assessment. We believe, however, that MR imaging will provide us with much more detailed and accurate information regarding tumor size, in addition to providing us with invaluable physiologic information not available by other means. To that end, each group will undergo baseline MR imaging following tumor ablation and then follow up MRI imaging x 2 following complete treatment. Group sizes were determined in consultation with our Statistician, Dr. David DeLong, to power the study to be able to detect a benefit based upon a halt in tumor growth or a delay in predicted tumor growth rate (based upon historical data with this tumor model) for the treatment groups relative to group A. We will use a total of 74 mice in total (including 4 for the establishment of an optimal RFA protocol as detailed below).Specific protocols for each of these steps have already been developed. With the exception of the RFA procedure, all protocols are well established in previous experiments. We will perform limited initial experiments in the previously mentioned animal model (n=4) to optimize the RFA procedure (choice of tip, time of ablation, etc.) and establish MRI parameters specific for the evaluation of RFA in these mice flank tumors. The Duke University Institutional Animal Care and Use Committee Protocol proposal detailing all of these protocols has already been approved (protocol # Clay A272-06-08).This project will be done through collaboration with the oncology lab of Dr. Michael Morse of the Department of Medicine. The immunotherapy protocol in mice and the immunoassays to be used are already well established in this laboratory's current work. This portion of the project will be supervised by Timothy Clay, PhD. The evaluation by MR perfusion and imaging would optimally be done in the Center for In Vivo Microscopy. This would be supervised by Laurence W. Hedlund, Ph.D. The RFA procedures will be performed by Anastasia Balius, MD and Rendon Nelson, MD, of the Department of Radiology. Overall management of the study will be directed by the partnership of Drs. Anastasia Balius and Timothy Clay.This is an elegant and well powered study design for the investigation of the complementary anti-tumor effect of RFA and tumor vaccine. Our hypothesis, if proven correct, has the potential to greatly expand the current role of RFA in the treatment of malignancy.
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