Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer

Hicham El Hadi, Imane Abdellaoui-Maane, Denise Kottwitz, Manal El Amrani, Nadia Bouchoutrouch, Zineb Qmichou, Mehdi Karkouri, Hicham ElAttar, Hassan Errihani, Pedro L. Fernandez, Youssef Bakri, Hassan Sefrioui, Abdeladim Moumen

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Abstract

Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods. HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%.

To our knowledge, this is the first study using the specific combination of RPL30 and RPL37 as reference genes for an accurate HER2 gene quantification in FFPE biopsy samples. Although further clinical validation regarding evolution and therapeutic response using RT-qPCR for the quantification of HER2 expression are still needed, the present study constitutes definitely a factual element that the RT-qPCR based assay may constitute a valid complementary test to accurately measure HER2 expression for a better treatment orientation. (C) 2017 Elsevier B.V. All rights reserved.

Original languageEnglish
Pages (from-to)114-122
Number of pages9
JournalGene
Volume605
DOIs
Publication statusPublished - 20 Mar 2017

Keywords

  • Breast cancer
  • HER2 expression
  • RT-qPCR
  • Endogenous control genes
  • Molecular diagnosis
  • FACTOR RECEPTOR 2
  • REVERSE TRANSCRIPTION-PCR
  • POLYMERASE CHAIN-REACTION
  • IN-SITU HYBRIDIZATION
  • QUANTITATIVE PCR
  • HOUSEKEEPING GENES
  • NORMALIZATION
  • HER-2/NEU
  • GUIDELINES
  • ASSAY

Cite this

El Hadi, H., Abdellaoui-Maane, I., Kottwitz, D., El Amrani, M., Bouchoutrouch, N., Qmichou, Z., Karkouri, M., ElAttar, H., Errihani, H., Fernandez, P. L., Bakri, Y., Sefrioui, H., & Moumen, A. (2017). Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer. Gene, 605, 114-122. https://doi.org/10.1016/j.gene.2016.12.027