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

Abstract

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selected reaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R 2 : 0.99 -1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website. MS based proteomics has traditionally been used to investigate complex biological systems, such as cell lines, plasma, or fresh-frozen tissues (1, 2). In the last decade however, MS proteomics has extended to the analysis of formalin-fixed paraffin-embedded (FFPE) 1 tissues (3). Formalin fixation is the gold standard for sample storage in clinical pathology because it allows optimal preservation of the morphological features of the tissue and it is economically attractive (storage at room temperature over several years or decades) (4). Sev-

Quantitative proteomic analysis of HER2 expression in the selection of gastric cancer patients for trastuzumab treatment

“…We have developed a HER2 SRM assay that measures a single peptide unique to HER2 and provides accurate quantitation of HER2 protein in a solubilized lysate prepared from laser microdissected tumor tissue [10]. Another laboratory has confirmed the ability of targeted proteomics to accurately quantitate HER2 and other oncogenic proteins in FFPE tissue [13]. …”

Peptide Selection for Targeted Protein Quantitation

“…Indeed, several previous works had successfully used peptides with missed cleavages or containing several potential sites of chemical modification for targeted proteomics analyses, as these peptides were shown to be reproducibly generated from replicate samples. [15][16][17] Therefore, given these evidences it seems advisable to not rely on the aminoacidic composition of a peptide but rather on experimental data-i.e. pilot shotgun or data-independent experiments with the system under study-to assess the quantitative behaviour of the peptides to be targeted, and thus select peptides that correctly represent true protein fold-changes in targeted proteomics studies.…”

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

“…The SRM assay showed good performance and high agreement with immunohistochemistry and FISH data. [145] The applications discussed above were selected to demonstrate the utility of targeted proteomic quantification as a powerful approach for biomarker verification with large sample sets.…”