J Breast Cancer.  2017 Sep;20(3):286-296. 10.4048/jbc.2017.20.3.286.

NanoString nCounter® Approach in Breast Cancer: A Comparative Analysis with Quantitative Real-Time Polymerase Chain Reaction, In Situ Hybridization, and Immunohistochemistry

• Hyeon J
• Cho SY
• Hong ME
• Kang SY
• Do I
• Im YH
• Cho EY

• KMID: 2438996
• DOI: http://doi.org/10.4048/jbc.2017.20.3.286

Abstract

PURPOSE
Accurate testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is essential for breast cancer treatment. At present, immunohistochemistry (IHC)/florescence in situ hybridization (FISH) are widely accepted as the standard testing methods. To investigate the value of NanoString nCounter®, we performed its comparative analysis with IHC/FISH and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the assessment of ER, PR, and HER2.
METHODS
Data on IHC/FISH results for ER, PR, and HER2 in 240 patients from a single tertiary hospital in Korea were collected and compared with NanoString nCounter® and qRT-PCR results at a single institution.
RESULTS
Expression levels for each gene using NanoString nCounter® showed good correlation with the corresponding data for protein expression by IHC (p<0.001) and gene amplification status for HER2 (p<0.001). Comparisons between gene expression and IHC data showed good overall agreement with a high area under the curve (AUC) for ESR1/ER (AUC=0.939), PgR/PR (AUC=0.796), and HER2/HER2 (AUC=0.989) (p<0.001).
CONCLUSION
The quantification of ER, PgR, and HER2 mRNA expression with NanoString nCounter® may be a viable alternative to conventional IHC/FISH methods.

Keyword

Breast neoplasms; ErbB-2; Gene expression; Immunohistochemistry; In situ hybridization

MeSH Terms

Breast Neoplasms*
Breast*
Estrogens
Gene Amplification
Gene Expression
Humans
Immunohistochemistry*
In Situ Hybridization*
Korea
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction*
Receptor, Epidermal Growth Factor
Receptors, Progesterone
Reverse Transcription
RNA, Messenger
Tertiary Care Centers
Estrogens
RNA, Messenger
Receptor, Epidermal Growth Factor
Receptors, Progesterone

Figure

• 

  Figure 1 Gene expression levels for ESR1, PgR, and HER2 by NanoString in comparison with corresponding protein biomarkers (immunohistochemistry [IHC]). Gene expression levels for ESR1 (A, D), PgR (B, E), and HER2 (C, F) using NanoString nCounter® had good correlation with the corresponding protein biomarkers (IHC) (p<0.001).ER=estrogen receptor; PR=progesterone receptor; HER2=human epidermal growth factor receptor 2.

• 

  Figure 2 Gene expression levels for HER2 by NanoString (A-C) and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) (D-F) in comparison with the gene amplification status by fluorescence in situ hybridization (FISH). Gene expression levels for HER2 using NanoString nCounter® had good correlation with the gene amplification status by FISH (p<0.001).HER2=human epidermal growth factor receptor 2; RQ=relative quantitation.

• 

  Figure 3 The estimation of overall agreements for gene expression levels compared with immunohistochemistry (IHC) results by receiver operator characteristic curve. Comparisons between the gene expression by NanoString and IHC data gave good overall agreement with a high area under the curve (AUC) for ESR1/ER (AUC=0.939) (A), PgR/PR (AUC=0.796) (B), and HER2/HER2 (AUC=0.989) (C) (p<0.001, respectively). The comparison between the gene expression by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) and IHC data gave also good overall agreement with a high AUC for HER2/HER2 (AUC=0.880) (D) (p<0.001).ER=estrogen receptor; PR=progesterone receptor; HER2=human epidermal growth factor receptor 2.

• 

  Figure 4 The general distribution of HER2 results of 240 specimens by FISH, IHC, and NanoString.HER2=human epidermal growth factor receptor 2; IHC=immunohistochemistry; FISH=fluorescence in situ hybridization; ND=not done; NA=data not applicable due to test failure; DCIS=ductal carcinoma in situ.

• 

  Figure 5 Correlation with NanoString counts for HER2 gene and relative quantitation (RQ) levels generated by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). (A) NanoString counts for HER2 gene showed high correlation with RQ levels generated by qRT-PCR (p<0.001). (B) Area under the curve of NanoString method is bigger than that of qRT-PCR method. The pairwise comparison of two receiver operator characteristic curves showed the statistically significant difference (p<0.001).HER2=human epidermal growth factor receptor 2.

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