Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time.
Author(s): Vassilakopoulou M, Parisi F, Siddiqui S, England AM, Zarella ER, Anagnostou V, Kluger Y, Hicks DG, Rimm DL, Neumeister VM
Publication: Lab Invest, 2015, Vol. 95, Page 334-341
PubMed ID: 25418580 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of cold ischemia time on the antigenicity of 12 phosphoproteins in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue.
Conclusion of Paper
Phospho-Tyrosine 4G10 and phospho-extracellular-signal-regulated kinases (Erk) 1/2 showed significantly decreased immunostaining with longer cold ischemia times. For phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTor), phospho-signal transducer and activator of transcription 3 (Stat3), phospho-stress activated protein kinase (SAPK), and phospho-Met, non-significant decreases in immunostaining were observed with increased cold ischemia time. Phospho-heat shock protein 27 (HSP27) immunostaining increased significantly while ribosomal protein phospho-S6 immunostaining showed a non-significant increase as cold ischemia time increased. Phospho-estrogen receptor (ER) and phospho-Janus kinase 2 (Jak2) immunostaining levels were not significantly affected by cold ischemia time.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of cold ischemia time on the antigenicity of 11 phosphoproteins in FFPE breast cancer tissue. A tissue microarray containing 93 breast cancer specimens (in duplicate) with a range of known cold ischemia times (25-415 min) was evaluated and quantitative immunofluorescence was performed using the Automated Quantitative Analysis (AQUA) method. Although initially included in the analysis, the authors report that statistical evaluation of phospho- human epidermal growth factor receptor 2 (Her2) staining was unable to be performed due to low numbers of Her2 positive cases and few specimens showing staining above the threshold to noise signal.
Summary of Findings:
Phospho-Tyrosine 4G10 and phospho-Erk1/2 showed significantly decreased immunostaining with longer cold ischemia times. For phosphor-AKT, phospho-mTor, phospho-Stat3, phospho-SAPK, and phospho-Met, non-significant decreases in immunostaining were observed with increased cold ischemia time. Phospho-HSP27 immunostaining increased significantly while ribosomal protein phospho-S6 immunostaining showed a non-significant increase as cold ischemia time increased. Phospho-ER and phospho-Jak2 immunostaining levels were not significantly affected by cold ischemia time.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Protein Immunohistochemistry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time <30 min
30-45 min
45-60 min
60-75 min
75-90 min
95-120 min
>120 min
Immunohistochemistry Specific Targeted peptide/protein Phospho-Her2
Phospho-ER
Phospho-Erk1/2
Phospho-Akt
Phospho-Stat3
Phospho-S6 Ribosomal Protein
Phospho-Jak2
Phospho-Met
Phospho-SAPK/JNK
Phospho-mTOR
4G10 Antiphosphotyrosine
pHSP27