Real Time RT-PCR Approach for the Evaluation of ERBB2 Overexpression in Breast Cancer Archival Samples: A Comparative Study With FISH, SISH, and Immunohistochemistry

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Abstract
We tested the reliability of real time reverse transcription polymerase chain reactions as an alternative method for the assessment of ERBB2 status in paraffin-embedded tissues of 83 patients with breast cancer and 20 non-neoplastic controls. PCR was also compared with the immunohistochemical (IHC) HercepTest score and with fluorescence (FISH) and silver (SISH) in-situ hybridization, in 42 selected cases. ERBB2 mRNA was overexpressed in 26/83 (31%) breast cancer samples, using a cutoff calculated as the mean value of the controls plus 3 SD or with the receiver operating curve. The PCR test showed a 96% sensitivity and a 100% specificity when compared with FISH, with an area under the receiver operating curve of 98.4%. Overexpression of ERBB2 at PCR was also significantly correlated with amplification in FISH (P<0.001, Mann-Whitney test) and in SISH (P<0.001, Mann-Whitney test), and with the IHC HercepTest scores 2 or 3 (P<0.001, Spearman rank correlation). FISH, SISH, and IHC were also compared with each other. ERBB2 amplification in FISH significantly correlated with that in SISH (P=0.002, chi test with a concordance of the 87%), but not with IHC HercepTest scores (P=0.214, chi test). Real time PCR is a reliable and cost-effective method for the assessment of ERBB2 status in archival breast cancer samples, compared with FISH. Its introduction in routine diagnostic pathology practice is feasible even if it requires amendments to the current clinical oncology protocols.