Immunohistochemistry versus PCR Technology for Molecular Subtyping of Breast Cancer: Multicentered Expereinces from Addis Ababa, Ethiopia

Abstract

The application of immunohistochemistry (IHC) for molecular characterization of breast cancer (BC) is of paramount importance; however, it is not universally standardized, subject to observer variability and quantifying is a challenge. An alternative molecular technology, such as endpoint reverse transcription (RT)-PCR gene expression analysis, may improve observer variability and diagnostic accuracy. This study was intended to compare IHC with the RT-PCR based technique and assess the potential of RT-PCR for molecular subtyping of BC. In this comparative cross-sectional study, 54 BC tissues were collected from three public hospitals in Addis Ababa and shipped to Gynaecology department at Martin-Luther University (Germany) for laboratory analysis. Only 41 samples were qualified for IHC and RT-PCR investigation of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 protein expression analysis. Kappa statistics was used to assess the concordance between the two techniques. The overall percent agreement between RT-PCR and IHC was 68.3% for ER (positive percent agreement [PPA] 71.1%; negative percent agreement [NPA] 33.3%), 39.0% for PR (PPA 14.3%; NPA 92.3%), and 82.9% for HER2 (PPA 62.5%; NPA 87.9%). Cohen's κ-values of 0.018 (< 0.20), 0.045 (< 0.200), and 0.481 (0.41-0.60) were generated for ER, PR, and HER2, respectively. Concordance for molecular subtypes was only 56.1% (23/41) and 0.20 kappa value. IHC and endpoint RT-PCR techniques have shown to be discordant for 43% samples. Molecular subtyping using endpoint RT-PCR was fairly concordant with IHC. Thus, endpoint RT-PCR may give an objective result, and can be applied for BC subtyping.

Keywords: Breast cancer; Immunohistochemistry; Molecular subtypes; Reverse transcription polymerase chain reaction.

Figure 1. Representative image of breast cancer tissue stained with haematoxylin and eosin (20×).

Arrows depict multinucleated cells with mitotic figures.

Figure 2. Representative images of IHC of ER, PR, HER2 and Ki-67 in human breast cancer tissue (20×).

Arrows showing abundantly stained areas for different receptors in breast cancer tissues. Breast cancer tissue was stained with antibodies specific for ER, PR, HER2 and Ki-67. Images depict ER-positive (A), PR-positive (B), HER2-positive (C) and Ki-67-positive (D). IHC, immunohistochemistry; ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2.

Figure 3. Gel document of endpoint RT-PCR.

(A) RPL37-A (reference gene); BT474: positive control, BT-20: negative control, P1-P4 patient samples, and NTC. (B) ESR-1 gene; BT474: positive control, BT-20: negative control, P1-P4 positive sample. (C) PGR gene; BT474: positive control, BT-20: negative control, P3 positive sample, P1, P2 and P4 negative sample. (D) ERBB2 gene; BT474: positive control, BT-20: negative control, P1-P4 negative sample. RT-PCR, reverse transcription PCR; NTC, no template control.