Microarray analysis of suppression subtracted hybridisation libraries identifies genes associated with breast cancer progression.

BACKGROUND: A major challenge of cancer research is to identify key molecules which are responsible for the development of the malignant metastatic phenotype, the major cause of cancer death. METHODS: Four subtracted cDNA libraries were constructed representing mRNAs differentially expressed between benign and malignant human breast tumour cells and between micro-dissected breast carcinoma in situ and invasive carcinoma. Hundreds of differentially expressed cDNAs from the libraries were micro-arrayed and screened with mRNAs from human breast tumor cell lines and clinical specimens. Gene products were further examined by RT-PCR and correlated with clinical data. RESULTS: The combination of subtractive hybridisation and microarray analysis has identified a panel of 15 cDNAs which shows strong correlations with estrogen receptor status, malignancy or relapse. This panel included S100P, which was associated with aneuploidy in cell lines and relapse/death in patients, and AGR2 which was associated with estrogen receptor and with patient relapse. X-box binding protein-1 is also an estrogen-dependent gene and is associated with better survival for breast cancer patients. CONCLUSIONS: The combination of subtracted cDNA libraries and microarray analysis has thus identified potential diagnostic/prognostic biomarkers and targets for cancer therapy, which have not been identified from common prognostic gene signatures.

Molecular analysis of a collection of clinical specimens stored at 4 degrees C as an alternative to snap-freezing.

It is critical for both basic and clinical translational cancer research to use high quality DNA, RNA and proteins from specimens with clinical outcome in order to validate novel diagnostic biomarkers and to monitor successful treatments for patients. However, using current standard procedures, the collection of specimens is often limited by the availability of liquid nitrogen in some hospitals and liquid nitrogen can be hazardous to transport. These problems would be eased if the tissue could be stored unfixed at 4 degrees C, conditions that are readily available in hospitals. Thus the effect of storing tissue specimens at 4 degrees C on the quality of DNA, RNA and protein has been examined. Clinical tissue samples were halved and kept either at 4 degrees C for up to 24 h or snap-frozen in liquid nitrogen within 30 min of removal from the patient. The results show that the quality of RNA, DNA and protein isolated from the specimens stored at 4 degrees C up to overnight is equal to that obtained from snap-frozen material. In conclusion, simplifying the collection procedure may allow for greater flexibility of conducting studies in units where liquid nitrogen is not readily available.