Cytological analysis of body fluids is currently used for detecting
cancer. The objective of this study was to determine if the herpes virus carrying an
enhanced green fluorescent protein (EGFP) could detect rare
cancer cells in body fluids against millions of normal cells. Human
cancer cells suspended with normal murine cells were infected with NV1066 at a multiplicity of
infection (MOI) of 0.5 and 1.0 for 18 h. Fluorescent microscopy and flow cytometry were used for EGFP detection of
cancer cells. EGFP-expressing cells were confirmed as
cancer cells with specific markers by immunohistochemistry staining. Limits of detection of
cancer cells in body fluid were measured by serial dilutions. Applicability of technique was confirmed with samples from patients with
malignant pleural effusions. NV1066 expressed EGFP in 111 human
cancer cell lines detected by fluorescent microscopy at an MOI of 0.5. NV1066 selectively infected
cancer cells and spared normal cells as confirmed by immunohistochemistry. Sensitivity of detecting fluorescent green cells was 92% (confidence interval [CI] 83% to 97%) at a ratio of 1
cancer cell to 1 million normal cells. EGFP-positive cells were detected by fluorescent microscopy in patients'
malignant pleural effusion samples. Our data show proof of the concept that NV1066-induced EGFP expression allows detection of a single
cancer cell against a background of 1 million normal cells. This method was demonstrated to be a reliable screening tool for human
cancer cells in a
suspension of normal murine cells as well as clinical specimens of
malignant pleural effusions.