title: Fluorescence-assisted cytological testing (FACT): Ex Vivo viral method for enhancing detection of rare cancer cells in body fluids.
abstract: 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.
authors.affiliation: Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
authors.forename: Prasad S
authors.initials: PS
authors.lastname: Adusumilli
authors.forename: Sepideh
authors.initials: S
authors.lastname: Gholami
authors.forename: Yun Shin
authors.initials: YS
authors.lastname: Chun
authors.forename: Michael
authors.initials: M
authors.lastname: Mullerad
authors.forename: Mei Ki
authors.initials: MK
authors.lastname: Chan
authors.forename: Zhenkun
authors.initials: Z
authors.lastname: Yu
authors.forename: Leah
authors.initials: L
authors.lastname: Ben-Porat
authors.forename: Valerie W
authors.initials: VW
authors.lastname: Rusch
authors.forename: Yuman
authors.initials: Y
authors.lastname: Fong
mesh_terms: D001826:Body Fluids / Q000378:metabolism*; D045744:Cell Line, Tumor; D003581:Cytodiagnosis / Q000379:methods*; D005434:Flow Cytometry; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D009369:Neoplasms / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D016066:Pleural Effusion, Malignant / Q000175:diagnosis / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D049452:Green Fluorescent Proteins