Putative Markets for the Detection of Breast Carcinoma Cells in Blood

By using the DF3 antigen (MUC1) as a molecular marker, previous workers reported the detection of tumour cell dissemination by reverse transcriptase Polymerase Chain Reaction (rt-PCR) in a pilot study of patients undergoing surgery for breast cancer (Brown et al., 1994). The aim of this study was to extend these findings and also examine the role of other epithelial markers for their suitability as molecular markers for detection of circulating breast carcinoma cells using rt-PCR. It was envisaged that this work could be extended to the examination of bone marrow and lymph nodes for occult metastases. RNA was prepared from MCF-7 breast carcinoma cells and peripheral blood leucocytes of healthy female volunteers. This RNA was screened for mRNA of MUC1, CK19 and CD44 (Exon 8-11) by rt-PCR and the results validated by Southern blots. Variable degrees of expression of MUC1 and CD44 (Exon 8-11) were detected in normal peripheral blood, rendering these genes non-specific for epithelial cells and therefore unsuitable for use as markers to detect circulating breast carcinoma cells. Although CK19 mRNA was apparently specific, it was deemed unsuitable for use as a marker of circulating breast cancer cells in light of its poor sensitivity. These findings prompted an alternative approach to this problem to continue the search for the ideal epithelial marker. Therefore, included in this work is RNA fingerprinting, a modified Differential Display by the PCR technique which is conceived to allow the identification, molecular cloning and sequencing of differentially expressed genes. This technique is devised to amplify mRNA and display their 3' termini on polyacrylamide gels. The aim of RNA fingerprinting is to identify and characterise the differentially expressed genes in breast cancer samples in contrast with normal blood. In this study a total of 18 differentially expressed genes (using limited primer combinations) were analysed and found to have variable degrees of background expression when used as potential markers. However, further work is required to characterise the differentially expressed genes using all other primer combinations. This study has cast major doubts on the validity of previously published studies in this area. Until a suitable epithelial marker for breast cancer is identified, further work to identify tumour cells in blood will not be feasible