DOUBLE-LABEL SUSPENSION IMMUNOHISTOCHEMISTRY FOR THE DETECTION OF GENE DYSFUNCTION IN SPUTUM

A method was developed to identify molecular markers and gene alterati ons in exfoliated cells present in preparations of sputum. A double-la bel immunocytochemistry procedure was performed in suspension, with an tibodies to cytokeratin intermediate filaments and to the p53 tumor su ppressor gene product. The procedure was carried out in sputum samples obtained from individuals with lung cancer, and p53 expression in exf oliated cells was correlated with p53 expression in corresponding lung tumors. An alkaline phosphatase substrate, which is readily discerned by light microscopy and enhanced by fluorescence at the FITC excitati on wavelengths, was used to detect immunolabeling by the monoclonal an tibody DO-7 to the nuclear protein p53. The epithelial cells in the sp utum were confirmed by fluorescence immunocytochemistry using a cockta il of monoclonal antibodies to a broad range of cytokeratins. Coexpres sion of cytokeratin and the p53 protein was detected in exfoliated cel ls with morphologies consistent with neoplasia and/or premalignant dis ease. These results demonstrate the feasibility of identifying gene al terations by immunocytochemistry in sputum from individuals with lung cancer. The detection of other gene alterations in individuals at risk for lung cancer using this method may enhance the benefit of traditio nal sputum cytology as a screening tool.