Comparison of IHC, FISH, SISH and qPCR methods in molecular diagnosis of breast cancer

Introduction: Her2 proto-oncogene amplification and protein overexpression is observed in 20–40% of patients with breast cancer and plays a crucial role in the biological behavior and pathogenesis of invasive breast cancer and its treatment. Both node-positive and node-negative breast cancer patients whose tumors exhibit Her2 amplification have a poor prognosis, an increased risk of recurrence and a high risk of disease-related death, showing an overall shorter survival time. However, it does predict a favorable response to chemotherapy and anti-Her2 antibody treatment. Therefore, it is very important to know the status of Her2 when thinking about biological therapy. The most common methods for testing of Her2 status are immunohistochemistry (IHC) for the detection of gene expression at the protein level, and fluorescence in situ hybridization (FISH) for the detection of gene amplification on the DNA level. In the present study we compare variable methods, which are based on different principles and each has its own advantages and disadvantages, to verify their dependability.

Materials and methods: We investigated samples from 131 female patients with invasive breast carcinoma. In all cases, the overexpression/amplification level of Her2 was determined by using manual immunohistochemistry (IHC) and/or automatic immunohistochemistry, fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH) and quantitative polymerase chain reaction (qPCR).

Results: Initially, we determined the Her2 status of 29 patients by IHC and qPCR analysis. After that we used qPCR and FISH methods in a group of 11 patients. In the next step, we performed analysis of Her2 status by manual and automatic IHC and FISH in another group of 35 patients. Finally, we analyzed another group of 67 patients by automatic IHC and SISH. We found a good correlation between IHC and qPCR, also between qPCR and FISH and between IHC and SISH. Moreover we found excellent correlation between manual and automatic IHC and between IHC and SISH.

Conclusion: By using variable methods we demonstrated possible ways for Her2 detection and their dependability. Our results proved that these methods are highly comparable for the detection of Her2 overexpression/amplification. As we have shown, quantitative PCR is a valuable tool for the evaluation of Her2 gene overexpression/amplification and correlates well with the results of IHC and FISH, respectively. Moreover, in contrast to IHC or SISH/FISH, the results obtained by quantitative PCR are not encumbered with any subjective error on the part of the evaluator.

This work was supported by Grant IGA MZ ČR NS/10575-3.