Real-time intraoperative detection of breast cancer axillary lymph node metastases using a green fluorescent protein-expressing herpes virus

OBJECTIVE: To investigate the use of a green fluorescent protein (GFP)-expressing oncolytic herpes virus to enable real-time intraoperative detection of breast cancer lymph node metastases. SUMMARY BACKGROUND DATA: Axillary lymph node status is the most important factor determining treatment, recurrence, and overall survival for women with breast cancer. The current methods of determining nodal status, however, have limitations. NV1066 is a novel oncolytic herpes viral strain that specifically infects cancer cells and expresses GFP. METHODS: Seven human breast cancer cell lines were infected in vitro with NV1066 and assessed for GFP expression, viral replication, and cytotoxicity. An in vivo model of breast cancer lymphatic metastasis was established in mice. Tumor-bearing mice were treated with NV1066 via injection into the primary tumor. Axillary lymph nodes were analyzed using an in vivo fluorescent imaging system. Histologic and molecular assessment of lymph nodes were performed using immunohistochemistry and reverse transcriptase PCR and operating characteristics were determined. RESULTS: NV1066 infected, expressed GFP, replicated within, and killed all human breast cancer cell lines in vitro. Injection of NV1066 into primary breast tumors resulted in viral transit to axillary lymph nodes, infection of lymphatic metastases, and GFP expression that was visualized with in vivo fluorescent imaging. Histologic and molecular confirmation demonstrated favorable operating characteristics of this method (sensitivity 80%; specificity 96%). CONCLUSIONS: We introduce a novel, sensitive, and specific method of lymphatic mapping that utilizes NV1066-guided cancer cell-specific viral production of GFP to enable real-time intraoperative detection of lymphatic metastases.