Progression-specific Genes Identified by Expression Profiling of Matched Ductal Carcinomas in situ and Invasive Breast Tumors, Combining Laser Capture Microdissection and Oligonucleotide Microarray Analysis
In breast cancer the progression of DCIS (ductal carcinoma in situ) to IDC (invasive ductal carcinoma) is a crucial step in developing metastasis–the major cause of mortality from malignant tumours. Understanding the molecular events accompanying this transition will be the basis (1) to improve the classification in high- and low-risk groups for DCIS patients according to risk of relapse or progression and (2) to advance in individualizing therapy for DCIS and IDC patients. In order to identify new prognostic markers and drug targets correlated with breast cancer progression, we combined Affymetrix microarray analysis with laser capture microdissection (LCM) to analyze matched DCIS and IDC tissue from nine patients. After extensive statistical analysis 548 Affymetrix probe sets were identified to be expressed differentially in either case of the nine tumors (446 up-regulated, 102 down-regulated, p-value <0.01), characterizing the transition from DCIS to IDC. Examples for known genes already associated with breast cancer invasion are e.g. PLAU/uPa and MMP11. Differential gene expression of 18 candidate genes was validated, analyzing triplicate real-time PCR experiments. Further 4 PCR-validated candidates were selected for in situ hybridization (ISH) experiments. Proteins of 5 PCR-validated candidates were detected using immunohistochemistry (IHC). Results of ISH and IHC confirm the localization for all transcripts to epithelial tumour cells. In conclusion, we identified progression-specific candidate genes using an accurate approach, combining LCM and microarray analysis. New candidates, not yet correlated with breast cancer progression, were evaluated using real-time PCR and transcript and protein could be localized to the epithelial tumour cells, respectively. Among these candidates are transmembrane receptors, transcription factors and tumour antigens, thus gene products with potential clinical importance.