ABSTRACT
BACKGROUND: We used a combination of genetic subtraction, silicon DNA microarray analysis, and quantitative PCR to identify tissue- and tumor-specific genes as diagnostic targets for breast cancer. METHODS AND RESULTS: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor-specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. CONCLUSION: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.
Subject(s)
Breast Neoplasms/blood , Breast Neoplasms/diagnosis , Breast Neoplasms/genetics , Neoplastic Cells, Circulating , Transcription, Genetic , DNA, Complementary/metabolism , Down-Regulation , Female , Humans , Magnetics , Mammaglobin A , Neoplasm Proteins/biosynthesis , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation , Uteroglobin/biosynthesisSubject(s)
Cell Separation/methods , Fetus/cytology , Pregnancy/blood , Avidin , Biotin , Female , Humans , Leukocyte Common Antigens/analysis , Male , Polymerase Chain Reaction , Y ChromosomeABSTRACT
We have employed RT-PCR of whole cells to develop a quantitative method for estimating the number of rare cells expressing a unique mRNA in a large, mixed population of cells. We have demonstrated that RT-PCR can be done on whole cells without the need for extraction of the RNA. This allows for a great saving of time and effort, as well as allowing quantitative analysis to be based on the total number of cells analyzed in a given aliquot and the presence or absence of the specific RT-PCR product. We have employed a limiting dilution series on whole cells, with multiple aliquots at each cell concentration to achieve more statistical power in the analysis of a rare cell type. We have used a nested amplification of the CD34 mRNA to be able to detect a single cell expressing the CD34 mRNA in a larger population of non-CD34-expressing cells. We demonstrate that by using this technique, cells from blood and bone marrow containing the CD34 mRNA can be followed quantitatively during a multistep purification involving immunoadsorption followed by fluorescence-activated cell sorting. We also demonstrate that many cells that express the CD34 protein on their surface no longer contain detectable levels of CD34 mRNA, a phenomenon that appears to be developmentally regulated.
