title: Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer.
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.
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco; Biochemistry and Immunology Laboratory, Faculty of Science, Rabat University Mohamed V, Avenue Ibn Battouta 1014, Rabat, Morocco. Electronic address: h.elhadi@mascir.com.
authors.forename: Hicham
authors.initials: H
authors.lastname: El Hadi
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco; Biochemistry and Immunology Laboratory, Faculty of Science, Rabat University Mohamed V, Avenue Ibn Battouta 1014, Rabat, Morocco. Electronic address: i.abdellaoui@mascir.com.
authors.forename: Imane
authors.initials: I
authors.lastname: Abdellaoui-Maane
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: denisekottwitz@web.de.
authors.forename: Denise
authors.initials: D
authors.lastname: Kottwitz
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: dahmani_manale@yahoo.fr.
authors.forename: Manal
authors.initials: M
authors.lastname: El Amrani
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: n.bouchoutrouch@mascir.com.
authors.forename: Nadia
authors.initials: N
authors.lastname: Bouchoutrouch
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: z.qmichou@mascir.com.
authors.forename: Zineb
authors.initials: Z
authors.lastname: Qmichou
authors.affiliation: Pathology Department, HOPITAL IBN ROCHD, Quartier Des Hopitaux, Casablanca, Morocco. Electronic address: mehdi.karkouri@menara.ma.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Karkouri
authors.affiliation: Laboratory of Pathology MlyIdriss I, Boulevard MoulayIdriss 1er Quartier Des Hopitaux, Casablanca, Morocco.
authors.forename: Hicham
authors.initials: H
authors.lastname: ElAttar
authors.affiliation: National Institute of Oncology (INO), Avenue Allal El Fassi, Rabat, Morocco.
authors.forename: Hassan
authors.initials: H
authors.lastname: Errihani
authors.affiliation: Department of Anatomical Pathology, Hospital Clínic and Institut d'Investigacions Biomèdiques August Pi iSunyer (IDIBAPS), University of Barcelona, Spain. Electronic address: plfernan@clinic.ub.es.
authors.forename: Pedro L
authors.initials: PL
authors.lastname: Fernandez
authors.affiliation: Biochemistry and Immunology Laboratory, Faculty of Science, Rabat University Mohamed V, Avenue Ibn Battouta 1014, Rabat, Morocco. Electronic address: ybakri@gmail.com.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Bakri
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: h.sefrioui@mascir.com.
authors.forename: Hassan
authors.initials: H
authors.lastname: Sefrioui
authors.affiliation: Medical Biotechnology Center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), Rue Mohamed Al JazouliMadinat Al Irfane, Rabat, Morocco. Electronic address: a.moumen@mascir.com.
authors.forename: Abdeladim
authors.initials: A
authors.lastname: Moumen
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D015870:Gene Expression; D020043:Genes, Essential; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D012015:Reference Standards; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D012269:Ribosomal Proteins; C484101:RPL37A protein, human; C508053:ERBB2 protein, human
keywords: Breast cancer; Endogenous control genes; HER2 expression; Molecular diagnosis; RT-qPCR