OBJECTIVE: To evaluate amplification of the HER-2/neu gene by fluorescence ir 
situ hybridization (FISH) in tumors with weakly positive (2+) 
immunohistochemical staining.
METHODS: A total of 1556 breast tumor biopsy specimens were referred to Mayo 
Medical Laboratories, Rochester, Minn, for HER2 testing between August and 
December 2000. Immunohistochemical (IHC) analysis was performed with use of a 
diagnostic test for the assessment of HER2 overexpression, the HercepTest. The 
IHC-stained slides were interpreted and scored on a scale ranging from 0 to 3+ 
according to Food and Drug Administration-approved guidelines. All specimens 
scored as 2+ were also routinely evaluated by FISH with use of a HER-2/neu DNA 
probe kit (PathVysion). Specimens were determined to be amplified if the ratio 
of HER-2/neu signals to chromosome 17 centromere (CEP17) signals was higher than 
2.0.
RESULTS: Thirty-eight percent of the specimens evaluated with the HercepTest 
were scored 0, 35% were 1+, 14% were 2+, and 13% were 3+. Of the 216 tumor 
specimens scored as 2+, 26 (12%) had a high level of HER-2/neu gene 
amplification, 54 (25%) demonstrated duplication of HER2, 4 (2%) deleted 
HER-2/neu and/or CEP17, and 123 (57%) had no apparent HER-2/neu anomaly, no 
apparent CEP17 anomaly, nor apparent single gain (aneusomy) of CEP17.
CONCLUSION: We recommend that all specimens with a 2+ HercepTest result be 
evaluated by FISH for HER-2/neu gene amplification. The results of both assays 
should be considered before making a decision to recommend anti-HER2 therapy.