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Comparison of immunohistochemistry and RT-qPCR for assessing ER, PR, HER2, and Ki67 and evaluating subtypes in patients with breast cancer

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Purpose

Currently, the most commonly applied method for the determination of breast cancer subtypes is to test estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 by immunohistochemistry (IHC). However, the IHC method has substantial intraobserver and interobserver variability. ESR1, PGR, ERBB2, and MKi67 mRNA tests by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay may improve the diagnostic objectivity and efficiency. Here, we compared the concordance between RT-qPCR and IHC for assessment of the same biomarkers and evaluated the subtypes.

Methods

A total of 265 eligible cases were divided into a training cohort and a validation cohort, and the expressions of ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 were tested by IHC and RT-qPCR. Then, the appropriate cutoff of RT-qPCR was calculated in the training cohort. The concordance between RT-qPCR and IHC was calculated for individual marker. In addition, we investigated the subtypes based on the RT-qPCR results.

Results

The Spearman correlation coefficients between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR were 0.768, 0.699, 0.762, and 0.387, respectively. The cutoff values for the RT-qPCR assay of ESR1 (1%), PGR (1%), ERBB2, and MKi67 (14%) were 35.539, 32.139, 36.398, and 29.176, respectively. The overall percent agreement (OPA) between ER/ESR1, PR/PGR, HER2/ERBB2, and Ki67/MKI67 by IHC and RT-qPCR was 92.48%, 73.68%, 92.80%, and 74.44%, respectively. A total of 224 (84.53%) specimens were concordant for the breast cancer subtypes (IHC-based type) by RT-qPCR.

Conclusion

Evaluation of breast cancer biomarker status by RT-qPCR was highly concordant with IHC. RT-qPCR can be used as a supplementary method to detect molecular markers of breast cancer.

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Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ESR1/ER:

Estrogen receptor alpha

PGR/PR:

Progesterone receptor

ERBB2/HER2:

Human epidermal growth factor receptor 2

MKI67/Ki67:

Marker of proliferation Ki67

mRNA:

Messenger ribonucleic acid

IHC:

Immunohistochemistry

FISH:

Fluorescence in situ hybridization

RT-qPCR:

Reverse transcription-quantitative polymerase chain reaction

FFPE:

Formalin-fixed paraffin-embedded

GOI:

Genes of interest

Cq:

Quantification cycle

REF:

Reference genes

Pc:

Positive control

ROC:

Receiver operating characteristics

OPA:

Overall percent agreement

PPA:

Positive percent agreement

NPA:

Negative percent agreement

PPV:

Positive predictive value

NPV:

Negative predictive value

CI:

Confidence intervals

FN:

False negative

FP:

False positive

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Acknowledgements

The authors would like to express our gratitude for those who have critically reviewed this manuscript and those who give us help during this experiment.

Funding

This work was supported by the National Key R&D Program of China (No. 2017YFC1308800), the National Natural Science Foundation of China (30900650, 81372501, 81572260, 81773299, 81701834, 81502327, 81172232, and 31430030), and the Guangdong Natural Science Foundation (2011B031800025, S2012010008378, S2012010008270, S2013010015327, 2013B021800126, 20090171120070, 9451008901002146, 2013B021800126, 2014A030313052, 2014J4100132, 2015A020214010, 2016A020215055, 201704020094, 2013B021800259, 2017B070705002, 16ykjc08, and 2015ykzd07).

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Author notes

  1. Lili Chen, Yanyang Chen, and Zhongpeng Xie have equal contribution as first authors.

Authors and Affiliations

  1. Molecular Diagnosis and Gene Testing Center, The First Affiliated Hospital, Sun Yat-sen University, No. 58, ZhongShan Second Road, Guangzhou, 510080, China

    Jiao Luo, Jianwen Zhou & Zunfu Ke

  2. Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Lili Chen, Yanyang Chen, Jiao Luo, Yuefeng Wang, Leilei Huang, Hongxia Li, Man Shu, Wenhui Zhang & Zunfu Ke

  3. Zhongshan School of Medicine, Sun Yat-sen University, No. 74, ZhongShan Second Road, Guangzhou, 510080, China

    Zhongpeng Xie

  4. Institute of Precision Medicine, Sun Yat-sen University, Guangzhou, China

    Zunfu Ke

  5. Beijing OriginPoly BioTec Co., Ltd, Beijing, China

    Linhai Wang & Pei Liu

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Contributions

ZFK, LLC, and YYC were involved in the conception of the study. LHW and PL performed the RT-qPCR assays; JL, YFW, JWZ, WHZ, and HXL provided study data and materials; YYC, ZPX, LLC, HXL, JL, MS, and LLH performed the statistical analysis and wrote the statistical plan; LLC, ZPX, YYC, LHW, and PL interpreted the data; LLC, YYC, and ZPX drafted the manuscript; ZFK and LLC critically revised the article, and all authors read and approved the final manuscript.

Corresponding author

Correspondence to Zunfu Ke.

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The authors have declared no conflicts of interest.

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All authors have read and approved the version of the manuscript.

Ethical approval

Approval for the study was granted by the Ethics Committee of First Affiliated Hospital of Sun Yat-sen University (No. 2016-032). All participants signed the consent.

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Chen, L., Chen, Y., Xie, Z. et al. Comparison of immunohistochemistry and RT-qPCR for assessing ER, PR, HER2, and Ki67 and evaluating subtypes in patients with breast cancer. Breast Cancer Res Treat 194, 517–529 (2022). https://doi.org/10.1007/s10549-022-06649-6

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  • DOI: https://doi.org/10.1007/s10549-022-06649-6

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