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

Reza Pazhoomand,Faranak Khodadad,Hossein Najmabad,Seyedeh Sedigheh Abedin,Masoud Karimlou,Atoosa Madadkar Sobhani,Farnaz Feiz,Alireza Iraniparast,Keivan Majidzadeh,Ideh Bahman,Fatemeh Aghakhani Moghadam,Elahe Keyhan,Farkhondeh Behjat,Ahad Muhammadnejad,Mehdi Banan

doi:10.7314/apjcp.2013.14.12.7621

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 still a 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). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy 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 that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were 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 MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRT- PCR is a prerequisite for determining the exact status of HER2.

Highlights

Breast cancer is responsible for 28% of the newly diagnosed cancer cases in women worldwide (Siegel et al, 2013)
Of the 113 invasive breast tumor samples included in this study, 93 samples were selected for further evaluation of human epidermal growth factor receptor 2 (HER2) status using MLPA, quantitative reverse transcription-PCR (qRT-PCR), and IHC
HER2 status by IHC and Fluorescence in Situ Hybridization (FISH) The ASCO/CAP scoring system for IHC was applied for interpretation of HercepTest results

Summary

Introduction

Breast cancer is responsible for 28% of the newly diagnosed cancer cases in women worldwide (Siegel et al, 2013). The treatment regimens for breast cancer patients are selected according to the biology and behavior of the tumor and depends on several factors such as: the human epidermal growth factor receptor 2 (HER2) status and the status of hormone (estrogen and progesterone) receptors. Despite the advances in targeted therapies, the disease recurs in approximately 30% of patients diagnosed with early stage breast cancer (Gonzalez-Angulo et al, 2007). The HER2 (ERBB2) proto-oncogene is located on chromosome 17 and encodes a 185-kD transmembrane tyrosine kinase, which has a critical role in determining patient prognosis and the treatment of breast cancer (O’Malley et al, 2001; Rosa et al, 2009). Recent studies have proposed a strong correlation between HER2 status and the expression of MED1, another gene that plays a critical role in anti-hormonal therapy resistance (Cui et al, 2012). Recent studies have proposed a strong correlation between HER2 status and the expression of MED1, another gene that plays a critical role in anti-hormonal therapy resistance (Cui et al, 2012). 7KHÀUVWOLQHWUHDWPHQWIRU+(5SDWLHQWVLV+HUFHSWLQ (trastuzumab), a humanized monoclonal antibody against

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