The classification and prognosis of breast cancers is complicated by many different breast cell types, morphological and genetic diversity among subtypes, and different developmental stages. Despite these complications, breast cancer patients with distinct molecular profiles are classified and treated as the same diagnostic and clinical prognostic group, which resulted in markedly different clinical outcomes. Therefore, a rapid, precise and cost-effective method to classify breast cancer subtypes and to determine progression stages by specific gene expression patterns is crucial for individual patient assessment and for personalized treatment. Recent research has examined many cell types in normal breast tissue, and found that myoepithelial cells and myofibroblasts display the most consistent changes between normal breast tissues and carcinomic ones. In addition, the genes that are expressed differentially primarily encode secreted and cell surface proteins, indicating large differences in cellular microenvironments between normal breast tissue and carcinomic tissue. The expression of more than 60 genes was observed in these cell lines using serial analysis of gene expression (SAGE), among them genes encoding chemokines CXCL12 and CXCL14. While CXCL12 has in the past been implicated in breast cancer metastasis, little is known about CXCL14. Further study on the epigenome in these types of cells has been examined by methylation-specific digital karyotyping (MSDK), and several other genes in breast cancer development have been implicated. While these findings are promising, methods like SAGE are time intensive, poorly suited for automation and cost intensive, which makes it poorly suited as a diagnostic tool in clinics. A novel method is being developed for more precise breast cancer diagnosis and molecular classification. This is based on our hypothesis that the diversity and progression of breast cancers might be accompanied by a corresponding diversity in gene expression patterns. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) provides a higher level of throughput for more accurate gene expression analysis of the breast cancers. MALDI-TOF MS has been extensively studied for DNA analysis, i.e., DNA sequencing, SNP genotyping, and allele frequency determination, since it is accurate, suitable for quantification and easy for automation. Previously SPC-SBE, a multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides (ddNTPs) and single base extension (SBE) reaction, has been developed for MALDI-TOF based genetic variation detection7. The method involves generating DNA fragments extended by a single base corresponding to the SNP of interest with SPC-ddNTPs such as biotinylated ddNTPs (biotin-ddNTPs). The DNA extension fragments containing biotin moiety at their 3' end are readily isolated from reaction mixture by utilizing a streptavidin-coated surface. The isolated DNA molecules are then analyzed with MALDI-TOF MS, allowing accurate DNA analysis for MALDI-TOF MS. We are exploring the use of biotin-ddNTPs and MALDI-TOF MS for gene expression analysis.Briefly, cDNAs will be reversely transcribed from mRNAs isolated from breast tissue (normal and cancerous) and the cDNAs of interest and a known amount of internal standard (internal standard 1, IS-1) will be PCR-amplified. A library of primers, one for each cDNA amplicon, will be extended using biotin-ddNTPs with another internal standard (IS-2). Extension products will be isolated by SPC on streptavidin-surface and analyzed with MALDI-TOF. Ratio between mass spectral peak areas will be used to determine the relative quantity of each transcript. The proposed system will enhance the accuracy in quantitative analysis of RNA samples, thus the level of gene expression. Additionally, the proposed approach will increase the number of gene transcripts that can be analyzed simultaneously. The proposed system utilizes biotin-ddNTPs and affinity capture of biotinylated DNA fragments. Development of a new method that assures rapid capturing, efficient washing and simple recovery of extension fragments is indispensable to further enhance the throughput provided by SPC. To this end, we will construct a novel microbead tip device for DNA isolation, which utilizes microbeads coated with biotin-binding proteins and a pipette tip. Together with the microbead tip system, the proposed system will provide higher level of throughput for accurate gene expression analysis.

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