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Breast Cancer Subtype Classification Using 4-Plex Droplet Digital PCR.

Wenwen Chen1, Jiaying Zheng1, Chang Wu2

  • 1Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Shenzhen University Health Science Center, Shenzhen, Guangdong, China.

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Summary

A new 4-plex droplet digital PCR (ddPCR) method accurately quantifies breast cancer biomarkers estrogen receptor 1 (ESR1), progesterone receptor (PGR), and human epidermal growth factor receptor 2 (HER2) in FFPE samples. This ddPCR assay shows high concordance with IHC, offering a reproducible alternative for breast cancer subtyping.

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Area of Science:

  • Molecular diagnostics
  • Biomarker analysis
  • Cancer research

Background:

  • Infiltrating ductal carcinoma (IDCA) is the most common invasive breast cancer subtype.
  • Immunohistochemistry (IHC) is standard for analyzing estrogen receptor 1 (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), but interpretations can be subjective and lack reproducibility.
  • Quantitative analysis of these biomarkers is crucial for guiding breast cancer treatment.

Purpose of the Study:

  • To develop and validate a 4-plex droplet digital PCR (ddPCR) assay for simultaneous, quantitative analysis of ESR1, PGR, ERBB2, and PUM1 gene expression.
  • To evaluate the sensitivity, reproducibility, and linear dynamic range of the developed ddPCR method.
  • To assess the concordance between the 4-plex ddPCR assay and IHC for classifying breast cancer subtypes using formalin-fixed paraffin-embedded (FFPE) samples.

Main Methods:

  • A 4-plex ddPCR assay was designed for simultaneous quantification of ESR1, PGR, ERBB2, and PUM1 expression levels.
  • The assay's sensitivity, reproducibility, and linear dynamic range were evaluated.
  • 95 FFPE samples from patients with breast IDCA were analyzed using the 4-plex ddPCR method.
  • Results from ddPCR were compared with conventional IHC staining for ER, PR, and HER2 status.

Main Results:

  • The limits of quantification (LOQ) for the ddPCR assay were established: 25 copies/reaction for ERBB2 and 50 copies/reaction for ESR1, PGR, and PUM1.
  • The assay demonstrated wide linear dynamic ranges, extending up to 1600 copies/reaction for all target genes.
  • High concordance rates were observed between 4-plex ddPCR and IHC: 96.8% for ERBB2, 91.5% for ESR1, and 85.1% for PGR.
  • Receiver operating characteristic (ROC) analysis yielded high areas under the curve (AUC): 0.991 for ERBB2, 0.977 for ESR1, and 0.920 for PGR.

Conclusions:

  • The 4-plex ddPCR assay provides a sensitive, reproducible, and quantitative method for analyzing key breast cancer biomarkers in FFPE samples.
  • The ddPCR assay shows high concordance with IHC, suggesting its potential as a more objective and reproducible alternative for breast cancer biomarker assessment and subtyping.
  • This quantitative approach can enhance the accuracy and reliability of breast cancer diagnosis and treatment stratification.