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Prospective Validation of a One-Step RT-qPCR-based Test for Quantifying HER2 Gene Expression in Breast Cancer
Basma Albanyahyati1,2,3, Hicham El Hadi1, Youssef Bakri2,3
1Diagnostic kit and medical devices center, Moroccan Foundation for Advanced Science Innovation and Research (MASCIR), University Mohammed VI Polytechnic, Lot 660, Hay Moulay Rachid Ben Guerir, 43150, Morocco.
Objective:
To Validate a one-step RT-qPCR as a reliable diagnostic tool in HER-2 positive breast cancer. Further, establishing this tool as a standard procedure to quantify HER-2 expression in Breast cancer patient.
Methods:
Here we report a prospective validation study that shows the concordance of one-step RT-qPCR in assessing the HER2 levels in formalin-fixed paraffin-embedded (FFPE) tissue samples with the current paradigm of diagnosis such as Immunohistochemistry (IHC) and Fluorescence-in-situ Hybridization (FISH). We collected 275 FFPE samples from the Department of Pathology, Ibn Rochd University Hospital Center. IHC was carried out in all the samples and those with a score of 2+ were also analyzed using FISH. We extracted mRNA from FFPE samples and performed RT-qPCR, and the results were compared with those obtained using IHC/FISH. HER2 mRNA levels were quantified and normalized using the reference genes RPL30 and RPL37, based on our earlier reports.
Results:
HER2 cut-off value was fixed at 11.954 corresponding to the combination of the best sensitivity and specificity (93.4% and 100%) respectively, and with positive predictive values PPV that reached 100% and a negative predictive value NPV that reached 89.4%. The results showed 100% concordance with FISH. The Kappa coefficient was 0.863 which indicated concordance with IHC. The area under the curve (AUC) which is an important parameter that determines the diagnostic accuracy of the test was calculated as AUC=0.955. The results were comparable to some of the recent studies published in a similar direction.
Conclusion:
Our study shows that one-step RT-qPCR-based quantitation is an accurate and reproducible test to record HER2 gene expression for a better treatment orientation focusing on maximal therapeutic and survival outcome.
