Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study.

Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.

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 SpectrumOrange 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 was 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.

Isolation and identification of metabolites of leukotriene A4 hydrolase inhibitor SC-57461 in rats.

The metabolic fate of SC-57461, N-methyl-N-[3-[4-(phenylmethyl)-phenoxy]propyl]-beta-alanine, a potent and specific inhibitor of the leukotriene A4 hydrolase, was determined by LC/MS/MS, NMR and GC/MS in male Sprague-Dawley rats. The major metabolites of SC-57461 in rats were the desmethyl metabolite, the hydroxylated metabolite, the N-oxide metabolite, the hydroxylamine metabolite, and the propionic acid metabolite. The N-oxide metabolite was found to be stable in the rat plasma and urine, but was unstable in most organic solvents (methanol, acetonitrile, and methylene chloride, etc.) because of the classic Cope reaction of the N-oxide, which led to the formation of the corresponding hydroxylamine product and acrylic acid. The hydroxylamine metabolite and acrylic acid were reactive in the biomatrix and could not be isolated in the in vivo samples. However, formation of the hydroxylamine metabolite and acrylic acid from the N-oxide metabolite in methylene chloride was verified by NMR. The propionic acid metabolite was found to be the common metabolite shared by SC-57461, N-oxide metabolite, as well as the hydroxylamine metabolite, which suggested a sequential metabolism of SC-57461 in rats. The ultimate fate of the propionic acid metabolite was incorporation into rat glycerolipid metabolism as a result of its structural similarity to aryl-substituted propionic acid, a known class of compounds that can be incorporated into rat glycerolipid metabolism. Finally, the isolated hydroxylated metabolite and the N-desmethyl metabolite were found to have excellent inhibitory effects toward leukotriene A4 hydrolase and therefore were the major active metabolites of SC-57461 in rats.

Determination of protein binding by in vitro charcoal adsorption.

Certain compounds such as SC-52151 have extensive nonspecific adsorption to the ultrafiltration devices or to dialysis membranes and therefore can not be measured by the conventional ultrafiltration or equilibrium dialysis methods. A new method based on charcoal adsorption was developed to overcome this difficulty. Unlike many conventional methods, which are based on the separation of free drug from bound drug under equilibrium conditions, the new method is operated under nonequilibrium conditions and involves measuring the time course of decline of the percentage of bound drug remaining in plasma while the free drug is being removed by charcoal adsorption. Theoretical aspects of the method and the data processing procedure are presented. SC-98A, a compound with minimal nonspecific adsorption to the ultrafiltration membrane, was used to demonstrate the applicability of this method against the ultrafiltration method. Using this method, the protein binding of SC-52151 in human plasma at 1.0 microgram/ml was determined to be in the range of 91.4-97.7% at room temperature.