title: Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time.
abstract: Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in post-translational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1-2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Vassilakopoulou
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Fabio
authors.initials: F
authors.lastname: Parisi
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Summar
authors.initials: S
authors.lastname: Siddiqui
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Allison M
authors.initials: AM
authors.lastname: England
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Zarella
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Valsamo
authors.initials: V
authors.lastname: Anagnostou
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Kluger
authors.affiliation: Department of Pathology, School of Medicine, University of Rochester, Rochester, NY, USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Hicks
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Rimm
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, CT, USA.
authors.forename: Veronique M
authors.initials: VM
authors.lastname: Neumeister
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D050377:Cold Ischemia; D000939:Epitopes / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods; D005557:Formaldehyde; D006801:Humans; D016612:Paraffin Embedding; D010750:Phosphoproteins / Q000378:metabolism*; D011379:Prognosis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods; D013997:Time Factors; D046888:Tissue Array Analysis; D016707:Tissue Fixation
chemical_list: D014408:Biomarkers, Tumor; D000939:Epitopes; D010750:Phosphoproteins; D005557:Formaldehyde