Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction

Gene amplification is one of the most important mechanisms leading to dereg ulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixtu re of nonneoplastic bystander and stroma cells. To overcome this obstacle a nd to develop an objective quantitative method we have combined laser-assis ted microdissection of tumor cells with the novel 5'-exonuclease-based real time polymerase chain reaction (PCR) assay. The latter method enables the h ighly reproducible exact quantification of minute amounts of nucleic adds. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase II alpha gene was determined in paraffin-embedded breast canc er specimens (n = 23) after immunohistochemical labeling and laser-based mi crodissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were s hown to escape from detection unless tumor cells were isolated by microdiss ection. In selected cases intratumor heterogeneity was demonstrated using a reas of similar to 50 to 100 cells. This novel approach combining immunohis tochemistry, laser microdissection, and quantitative kinetic PCR allows mor phology-guided studies in archival tissue specimens and will enable the exa ct quantification of gene copy numbers in even small and precancerous lesio ns.