Reproducibility of LSI HER-2/neu SpectrumOrange and CEP 17 SpectrumGreen Dual Color deoxyribonucleic acid probe kit. For enumeration of gene amplification in paraffin-embedded specimens: a multicenter clinical validation study.

Overexpression and/or amplification of HER-2/neu gene have been found to be prognostic and predictive in breast and other cancers. Fluorescence in situ hybridization (FISH) assay, with its sensitivity and specificity, can be a superior method of detection when its performance characteristics are demonstrated. A multicenter study was initiated to evaluate the reproducibility of the LSI HER-2/neu Spectrum-Orange and CEP 17 SpectrumGreen Dual Color DNA Probe for enumeration of both the HER-2/neu gene and chromosome 17 (signals) in interphase cells. Section slides were prepared from four cell lines (H, E, R, and N) with known ratios of the HER-2/neu to CEP 17 copy numbers (approximately H = 1.10, E = 1.70, R = 4.50, N = 9.0). The study variable was the ratios of the HER-2/neu to chromosome 17 copy numbers. Reproducibility with respect to assay, site, lot, day and reader was evaluated at 3 centers. Out of 120 specimen slides, 100 percent were successfully assayed. There were no significant differences among: (1) four repeated assays of the same specimen (p = 0.99), (2) the four probe lots (p = 0.33), or (3) the four study days (p = 0.54). There were statistically significant, but not important differences among centers and between readers. The ratios of the HER-2/neu to chromosome 17 copy numbers were estimated with accuracy and precision; the mean ratios (and sd) for specimens, H, E, R, and N were 1.05 (0.06), 1.81 (0.12), 4.48 (0.28), and 8.60 (1.23), respectively. In summary, assays with the LSI HER-2/neu and CEP 17 Dual Color DNA Probe Kit, conducted at three sites by 6 different technicians, over 8 assay days, using kits from four lots, were performed with a high success rate in paraffin-embedded specimens. The signal enumeration was also accurate and precise. This study demonstrated that the results obtained by using the LSI HER-2/neu SpectrumOrange and CEP 17 SpectrumGreen Dual Color DNA Probe Kit are reliable and reproducible.