Borderline HER‐2 breast cancer cases: Histochemical versus real‐time PCR analysis and impact of different cut‐off values

Abstract

Seventy‐one cases that had resulted borderline for HER‐2 protein expression at conventional immunohistochemical assay (2+) were assessed for HER‐2 gene amplification by real‐time PCR and by FISH in accordance with the manufacturer's recommendations (gene amplification with ratio ⩾2 in both methods). Thirty‐three out of 71 cases (47%) resulted amplified at real‐time PCR analysis, whereas 15 cases resulted positive at FISH (21%). Apparently, PCR was more sensitive than FISH in HER‐2 determination, only 10 cases resulting amplified in both tests. When the mean ratio value obtained in all PCR experiments was adopted as threshold in determining HER‐2 gene amplification, the apparent sensitivity of PCR was reduced but correlation between PCR and FISH results was dramatically increased. Furthermore, when the mean PCR ratio value observed in the FISH‐positive group was chosen as threshold, the best agreement between PCR and FISH results was achieved. Therefore, we found that the proposed threshold ratio value of ⩾2 is not accurate in separating HER‐2 amplified and non‐amplified cases. We suggest that the threshold ratio value in PCR tests should be determined in each laboratory using FISH controlled cases. Finally, above certain in‐lab generated threshold values, PCR might be proposed as a highly predictive positive test in HER‐2 assessment.

• FISH
• immunohistochemistry
• interassay correlation
• LightCycler
• test validation
• test variability
• threshold value

Acknowledgements

The costs of this analysis were covered in part by Roche Diagnostics Italian Division. Special thanks to Antonio Lavelli, Field Applicative Specialist (Roche), for his technical support. The work was also supported by the Italian Ministry for University, Research and Scientific Technology (MURST), grant no. 7020014.