Introduction:Cytological detection of free peritoneal cancer cells detected in gastric cancer patients offers important prognostic information and may affect staging and treatment. However, conventional cytology by Papanicolaou staining clearly does not detect all cases of peritoneal disease. We evaluated a novel technique for detecting free peritoneal gastric cancer cells using Newcastle Disease Virus (NDV-GFP), a non-pathogenic virus containing the enhanced green fluorescent protein (GFP) gene.Methods:NDV-GFP was tested upon MKN-1 human gastric adenocarcinoma cells plated against a background of normal rat hepatocytes to determine tumor-specific viral infection and GFP expression. A clinical sample of malignant ascites was then processed, incubated with increasing doses of virus, and evaluated with fluorescence microscopy for optimal NDV-GFP dose determination. Peritoneal lavage samples from 22 patients with biopsy-proven gastric adenocarcinoma undergoing staging laparoscopy were then evaluated with NDV-GFP. Green fluorescent cells were further molecularly characterized.Results: NDV-GFP at a dose of 5E6 PFU specifically infects MKN-1 gastric adenocarcinoma cells and can detect 1 cancer cell against 1 million benign rat hepatocytes. GFP expression was seen at 6 hours from infection and was detectable for over 24 hours. NDV-GFP at a dose of 5E4 plaque forming units (PFU) produced detectable GFP expression in a clinical sample of malignant ascites, which was enhanced with higher viral doses. For practicality, further samples were infected with 5E6 PFU. Non-cancerous cells, such as red blood cells, were found to be non-GFP expressing. GFP- expressing cells counterstained positive for CEA expression, confirming their cancerous origin. GFP expression was seen in lavage samples from all 22 patients, while cytology was positive in only 5 of these patients. While 9/22 (40.9%) patients were stage IV with M1 disease, only 5/9 (55.6%) were positive by cytology. In contrast, all 9 samples were positive by NDV-GFP detection. Furthermore, when evaluating tumor size, GFP expression was markedly enhanced in T3 disease, detecting 16 patients with T3 tumors versus 2 patients detected by cytology. When considering nodal status, NDV-GFP detected 10 N1 patients compared to no N1 patients detected by cytology.Conclusions: NDV-GFP specifically targets and infects gastric cancer cells. NDV-GFP enhances detection of free peritoneal cancer cells in gastric cancer patients and offers a more rapid and sensitive diagnostic tool compared to conventional cytology. This novel diagnostic modality may offer important prognostic information.