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Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable
Maria Vassilakopoulou1, Fabio Parisi1, Summar Siddiqui1
1Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
Laboratory Investigation; a Journal of Technical Methods and Pathology
|November 25, 2014
Summary
Cold ischemic time significantly impacts biomarker assessment in breast cancer tissues. Some phosphoepitopes are labile, necessitating strict quality control for accurate targeted therapy decisions.
Area of Science:
- Oncology
- Biomarker Research
- Pathology
Background:
- Accurate biomarker assessment is crucial for individualized cancer therapies.
- Preanalytical variables, like delayed formalin fixation, can significantly affect biomarker levels.
- Phosphorylated proteins are particularly susceptible to degradation during cold ischemic time.
Purpose of the Study:
- To evaluate the impact of cold ischemic time on the antigenicity of key phosphoprotein biomarkers in breast cancer specimens.
- To identify which phosphoepitopes remain stable and which are labile under varying fixation delays.
Main Methods:
- Utilized a tissue microarray of 93 breast cancer specimens with documented time-to-fixation.
- Quantified changes in 12 phosphoepitopes using AQUA technology for quantitative immunofluorescence.
- Performed univariate linear regression and bootstrapping for statistical analysis.
Main Results:
- Most tested phosphoepitopes showed altered expression levels with increasing cold ischemic time.
- Certain phosphoproteins (e.g., phospho-HSP27, phospho-S6 RP) increased in levels.
- Several labile phosphoproteins (e.g., phospho-AKT, phospho-ERK1/2) lost antigenicity within 1-2 hours of cold ischemic time.
Conclusions:
- Cold ischemic time is a critical preanalytical variable affecting phosphoprotein biomarker reliability in breast cancer.
- Strict monitoring and quality control of specimen collection are essential for accurate biomarker measurement.
- A few phosphoepitopes (phospho-JAK2, phospho-ER) demonstrate robustness for companion diagnostics.
