Quantitative PCR – new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real‐time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization

Abstract

Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real‐time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Tumor biopsies were collected from 112 patients diagnosed with early breast cancer from January 1990 to March 1994. The samples were analyzed for HER2 DNA amplification by real‐time PCR on LightCycler and by FISH and for HER2 protein expression by IHC. Inter‐assay variation for HER2 measured by LightCycler was 10% (×=3.1; n=17). Amplification ≥2 was observed in 19% of the patients. Concordance rates between real‐time PCR and the other methods were 91% (IHC) and 92% (FISH). The correlation between real‐time PCR and FISH was highly significant (p<0.001). The “LightCycler‐HER2/neu DNA quantification kit” produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real‐time PCR compares with FISH and IHC. The data show that results obtained for amplification of HER2 by real‐time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH.

• Breast cancer
• epidermal growth factor system
• HER2
• quantitative PCR
• real time PCR