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    Fluorescent Imaging To Delineate Margins In Breast Conservative Surgery Using Green Fluorescent Protein Expressing Oncolytic Herpes Virus

    Scientific Abstract:
    Title: Fluorescent Imaging To Delineate Margins In Breast Conservative Surgery Using Green Fluorescent Protein Expressing Oncolytic Herpes Virus Background: In breast conservation therapy (BCT), a positive margin is associated with a higher local recurrence rate. With increasing detection of nonpalpable lesions by mammography, surgeon and pathologist are faced with the challenge of completely excising tumor while minimizing removal of surrounding normal tissue. The amount of tissue required to assess margins for mammographically detected carcinoma is often more extensive than that needed for palpable disease. Current methods of frozen section analysis with ink margins is limited by the freezing artifacts, cost, limitation of amount of tissue left for permanent section, and sampling inadequacies that may result in possible need for subsequent re-excision. Furthermore, microscopic residual cancer entrapped in the relatively hypoxic environment of a margin-positive lumpectomy scar might be resistant to chemo- and radiation therapy. Imaging techniques that aid in complete primary excision of tumors may improve outcome of BCT. Oncolytic herpes simplex viruses (HSV) have been constructed that are highly selective in infection, replication within, and killing of cancer cells. One such mutant, NV 1066, carries a transgene for green fluorescent protein (GFP) and expresses green fluorescence in infected cells. This proposal seeks to determine if NV1066 1) can be used to rapidly and specifically image breast cancer to delineate surgical margins, and 2) can kill such infected tumor cells. Objectives and Specific Aims: 1) To determine the infectivity, replication, and cytotoxicity of NV1066 in breast cancer cell lines with various growth and physical characteristics in vitro and in vivo. 2) To determine the time course of GFP expression after exposure of breast cancer cells or non-malignant adipose tissue to NV1066. 3) To determine the detection sensitivity of NV1066 mediated fluorescent imaging (with and without automated detector) in delineating the tumor margin compared to traditional histology, 4) To determine the possible interference of operative techniques (electrocautery, blue dye and routine histological staining) with GFP detection, and 5) To determine the influence of GFP-guided surgical excision on local recurrence and survival difference. Study Design: In vitro, infectivity, GFP expression intensity, viral replication and cytotoxicity will be tested against several breast cancer cell lines. In vivo studies will be conducted with rat and mouse breast cancer tumor models. Tumors will be implanted in either the flank or in the retro-peritoneal fat for these studies. Tumors of various sizes will be studied ranging from barely palpable to large. At various time points after viral injection, tumor will be completely excised and assessed for surface GFP expression or deep GFP appearance using detectors with fixed excitation (470±40nm) and emission (500 nm) filters to accommodate a GFP minor excitation peak of 475 nm and an emission peak of 509nm. Surface expression will be used to assess margin while deep detection will help assess for ability of this technique for guiding identification of tumor site. GFP expression intensity to background ratio will be assessed by direct measurement and by automated detection using a gated detector. GFP detection will be compared to histology, to immuno-staining for tumor and virus, and to PCR for tumor and virus (cytokeratin and ICP0 respectively). Potential benefits and Outcomes to research: If HSV mediated GFP imaging proves feasible in these preclinical studies, it would lend support to clinical studies that may guide the surgeon in identification of unpalpable tumors and improve the likelihood of achieving a negative margin at time of primary surgical therapy, which have as its goals improved survival and better post-operative cosmetic results. Furthermore, oncolytic virus being therapeutic and not just a imaging marker may also replicate and kill residual cancer cells. The lessons learnt from this project can potentially be applied for any other tumor margin identification. Lastly, viruses similar to NV1066 are already in human therapeutic trials, making transition to human trials for any encouraging findings readily possible.

    Lay Abstract:
    Title: Fluorescent Imaging To Delineate Margins In Breast Conservative Surgery Using Green Fluorescent Protein Expressing Oncolytic Herpes Virus The goals of breast conservation surgery are complete excision of locally detectable tumor while minimizing loss of surrounding non-malignant breast tissue. For non-palpable breast cancer, the surgeon and pathologist cannot directly visualize the border between tumor cells and normal surrounding tissue in the operating room. Currently, after the surgeon removes the suspected lesion, which is marked by the radiologist, the pathologist examines the tissue by painting the margins with ink and assessing the closeness of the cancer to the margin. This method takes 3-5 days to accurately report back the negativity of the margins. If a sufficient margin is not removed during the initial surgery, there is a substantially increased chance of recurrence, resulting in additional surgeries and even possible need to remove the entire breast. While this risk is thought to be reduced by removing more tissue, cosmesis can be unacceptably impacted by more extensive surgery. One promising new approach for the treatment of solid tumors such as breast cancer is to modify well-characterized human herpes viruses to specifically kill tumor cells. These viruses selectively replicate in cancer cells resulting in lysis of the cell releasing new viruses that can spread to the surrounding tumor. One of these viruses being studied in our laboratory is modified to carry a gene that expresses a green fluorescent protein (GFP) in the infected cancer cell. This virus is useful not only to kill cancer, but also as a diagnostic tool in the early detection of small focus of cancer that can not be visualized by the naked eye or by microscopy without special staining. In this research proposal, we specifically will examine: 1) the ability of virus to infect different types of breast cancer cells, 2) emission of green fluorescent protein in tumors with relation to varying doses and time points, 3) ability to detect the greenness using an automated detection system, and 4) possible interference of green fluorescent protein with routine histological stains and techniques. If found to be of use in animal models, we anticipate that in humans, the GFP expressing herpes virus can be injected while the patient is undergoing a radiological intervention procedure to localize a non-palpable tumor. At surgery, after the specimen is removed, the pathologist can examine the specimen under a special microscope to look for green fluorescence and informs the surgeon about the adequacy of the margins, while the patient is still in the operating room. If there is evidence of cancer, the surgeon can excise that area precisely (with the aid of a telescope fitted with fluorescent filters) avoiding unnecessary removal of big portions of breast. The spin-offs of this gene therapy imaging technology is that even if some cancer cells escape surgical removal, the oncolytic virus being therapeutic, not just a color marker, replicates and kills those left-out cancer cells. As the virus is injected locally and as almost the entire virus injected comes out with the specimen, the potential side effects of systemic injections can be avoided. If our current research proposal of advancing the tumor margin detection from the age of painting ink to gene therapy era is proven to be feasible, rapid transition to human trials is possible, thus allowing a more timely assessment of complete tumor excision, which will lead to improved survival and better post-operative cosmetic results.