Exact quantification of gene amplification in archival tissue sections by laser-assisted microdissection and real-time PCR

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Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is hampered by a mixture of non-neoplastic bystander cells. In order to overcome this shortcoming and to develop an objective quantification method, we have combined laser-based microdissection of tumor cells with the novel 5'-exonuclease based real-time PCR-assay that enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system, amplification of the c-erb-B2/Her-2/neu gene and the adjacent topoisomerase IIα gene were determined in paraffin-embedded breast cancer tissue (n = 23) after immunohistochemical labelling and laser-based microdissection.

The quantitative assay was linear over a broad range approaching the theoretical detection limit. 91% (21/23) of the specimens were suitable for the PCR analysis. The immunohistochemical labelling of cells did not interfere at all with the quantitative PCR. 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. In selected cases intratumor heterogeneity was analysed using areas of approx. 50-100 cells. In addition, we have already started the systematic analysis of gene amplification in DCIS of the breast to correlate morphological classification systems with the results of molecular analysis.

This novel approach, combining immunohistochemistry, laser-microdissection and quantitative kinetic PCR, allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers even in small and precancerous lesions.