Detection of HER2 Status in Breast Cancer: Comparison of Current Methods with MLPA and Real-time RT-PCR

Abstract

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer.There is no globally accepted method for determining its status, and which method is most precise is stilla matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR(qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). Inparallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess theaccuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization(FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results thatdisagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four sampleswere not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these sampleswere confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973,respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was(0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPAtechniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlationsbetween MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, wepropose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCRis a prerequisite for determining the exact status of HER2.