Transcriptional complementarity in breast cancer: application to detection of circulating tumor cells.

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.

Expression cloning of an immunodominant family of Mycobacterium tuberculosis antigens using human CD4(+) T cells.

Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) is likely to be dependent on the identification of T cell antigens that induce strong proliferation and interferon gamma production from healthy purified protein derivative (PPD)(+) donors. We have developed a sensitive and rapid technique for screening an Mtb genomic library expressed in Escherichia coli using Mtb-specific CD4(+) T cells. Using this technique, we identified a family of highly related Mtb antigens. The gene of one family member encodes a 9.9-kD antigen, termed Mtb9.9A. Recombinant Mtb9.9A protein, expressed and purified from E. coli, elicited strong T cell proliferation and IFN-gamma production by peripheral blood mononuclear cells from PPD(+) but not PPD(-) individuals. Southern blot analysis and examination of the Mtb genome sequence revealed a family of highly related genes. A T cell line from a PPD(+) donor that failed to react with recombinant Mtb9.9A recognized one of the other family members, Mtb9.9C. Synthetic peptides were used to map the T cell epitope recognized by this line, and revealed a single amino acid substitution in this region when compared with Mtb9.9A. The direct identification of antigens using T cells from immune donors will undoubtedly be critical for the development of vaccines to several intracellular pathogens.

Quantitative analysis of CD34+ stem cells using RT-PCR on whole cells.

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.