Evaluation of circulating tumour cells expressing CD44 variants in the blood of gastric cancer patients by flow cytometry

The occurrence of circulating tumour cells in the blood of 51 patients with gastric cancer (stages I-IV) was studied using flow cytometry, cell sortin g, immunohistochemistry and reverse trascription polymerase chain reaction (RT-PCR). The lysed whole blood samples were stained with monoclonal antibo dies against common leukocyte antigen (CD45), epithelial membrane antigen ( EMA), tumour associated glycoprotein 72kD (TAG72) CD44 variants (nu 5 and n u 6) and analysed by flow cytometry within ungated or CD45(-) gated populat ions. The frequency of detection of TAG72 CD44 nu 5(+) and nu 6(+) cells wi thin CD45(-) gate was considerably increased in comparision to the ungated population. Furthermore, the presence of tumour cells was directly demonstr ated by immunostaining for cytokeratin 18 of sorted CD45- population. The p resence of CD44 nu 5(+), nu 6 cells and CD44 nu-mRNA in the blood was compa red to their expression in the primary tumour. The occurrence of circulatin g CD44 nu(+) cells was associated with their presence in the primary tumour and CD44 nu-mRNA: in the blood. The method described may provide a sensiti ve tumour marker-independent tool for detection of circulating tumour cells in cancer patients.