Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

Area of Science:

• Biochemistry
• Proteomics
• Cancer Research

Background:

• Accurate protein quantification in formalin-fixed paraffin-embedded (FFPE) tissues is crucial for biomarker discovery.
• Targeted mass spectrometry offers a promising approach for precise protein measurement in FFPE samples.

Purpose of the Study:

• To develop and validate a selected reaction monitoring (SRM) assay for quantifying the human receptor tyrosine-protein kinase erbB-2 (HER2) protein in FFPE breast tumors.
• To assess the assay's analytical performance and its ability to correlate with established HER2 amplification detection methods.

Main Methods:

• Identified HER2 peptide candidates using untargeted mass spectrometry in cell lines.
• Developed a multiplexed SRM assay for six optimal peptides, evaluating linearity, precision, and lower limit of quantification (LLOQ).
• Quantified HER2 peptides in 40 FFPE breast tumor tissues and compared results with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) data.

Main Results:

• The SRM assay demonstrated excellent linearity (R(2): 0.99-1.00) and a low LLOQ (0.155 fmol on column) for all evaluated peptides.
• Quantified HER2 peptide levels were highly correlated, serving as reliable surrogates for total protein amounts in FFPE tissues.
• SRM measurements correctly predicted 90% of HER2 amplification cases, showing high agreement with IHC and FISH results.

Conclusions:

• The developed SRM assay exhibits robust analytical performance for quantifying HER2 protein expression in FFPE breast cancer tissues.
• This targeted mass spectrometry approach is a powerful tool for biomarker discovery and has high concordance with standard clinical pathology methods.
• The study validates SRM as a reliable method for accurate protein quantification in FFPE samples, supporting its use in clinical research.