An Optimized Droplet Digital PCR Assay for HER2 Copy Number Variation in Breast Cancer Based on Multi-reference Genes

Area of Science:

• Oncology
• Molecular Diagnostics
• Genetics

Background:

• Human epidermal growth factor receptor 2 (HER2) over-expression occurs in 15-30% of invasive breast cancers, necessitating targeted therapy.
• Current HER2 diagnostic methods (IHC, FISH) involve manual interpretation, leading to subjectivity and potential inaccuracies.
• Accurate HER2 status assessment is critical for guiding effective breast cancer treatment decisions.

Purpose of the Study:

• To develop and validate a precise and objective droplet digital PCR (ddPCR) assay for assessing HER2 amplification status in formalin-fixed paraffin-embedded (FFPE) breast cancer samples.
• To compare the diagnostic performance of the novel ddPCR assay against existing clinical methods.

Main Methods:

• Development of a four-fluorescence ddPCR assay utilizing multiple reference genes.
• Validation of the ddPCR assay on 101 clinical breast cancer FFPE samples.
• Comparison of ddPCR results with clinicopathological data, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR).

Main Results:

• The ddPCR assay using two reference genes demonstrated high sensitivity (82.6%), specificity (98.7%), and consistency (95.0%) in determining HER2 status.
• Whole genome sequencing confirmed the superior accuracy of the ddPCR assay compared to IHC, FISH, and qPCR.
• The ddPCR assay effectively reduced errors associated with chromosome 17 abnormalities.

Conclusions:

• The multi-reference gene ddPCR assay significantly enhances the accuracy of HER2 status detection in breast cancer FFPE samples.
• This ddPCR method shows promise as a reliable alternative or complementary tool to conventional IHC and FISH testing.
• The assay's feasibility for both tissue and liquid biopsies broadens its potential clinical applications.