Dual-colour chromogenic in situ hybridization for testing of HER-2 oncogene amplification in archival breast tumours

Chromogenic in situ hybridization (CISH), which uses an enzymatic reaction to detect the hybridized DNA probe, is a new alternative to fluorescence in situ hybridization (FISH) for the assessment of HER-2 oncogene amplification status in breast cancer. The main advantage of CISH over FISH is the use of bright-field microscopy, which is rapid and allows the histopathological evaluation of tumour tissue sections. The main disadvantage of CISH has been the use of a single probe, thereby making it necessary to hybridize the control probe (chromosome 17 centromere) on an adjacent tissue section. The present paper presents an efficient protocol for dual-colour CISH (dc-CISH) based on the co-hybridization of probes to the HER-2 oncogene and chromosome 17 centromere. The probes were detected sequentially with antibodies to digoxigenin and biotin and with secondary antibody polymers labelled with horseradish peroxidase and alkaline phosphatase. The peroxidase reaction was visualized with tetramethyl benzidine (green reaction product) and the alkaline phosphatase reaction with New Fuchsin (red reaction product). The accuracy of the method was verified by comparing the results for four cell lines and 40 tumour samples with those obtained using FISH (Vysis Inc.). The results of FISH and dc-CISH showed high concordance (91%, Kappa coefficient = 0.82). It is concluded that dual-colour CISH, which is a new alternative to FISH enables the assessment of copy number ratio (HER-2/17 centromere) in conjunction with proper histopathological evaluation and the ease of bright-field microscopy.