Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables

Abstract

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, to that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Antibodies to phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, Estrogen Receptor, Ki67, cytokeratin and GAPDH were measured. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared to biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, Estrogen Receptor and cytokeratin showed significant loss of antigenicity. This data suggests that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time

“…Furthermore our study was performed on a single cohort. However, similar trends of loss of phosphoepitopes have been reported previously, 18,20 None the less, validation on another cohort could strengthen these results. Also, the trends seen here may become statistically stronger with larger sample sizes and other patient cohorts.…”

“…15 Similarly elevated phospho-S6 kinase-1 and phospho-AKT levels are said to predict resistance to neoadjuvant chemotherapy 16 Recent studies have emphasized the importance of tissue handling and standardization of preanalytical variables as these can have dramatic impact on the quality of harvested tissue and therefore on protein expression levels. [17][18][19][20][21][22][23] This often requires special attention to tissue handling avoiding modification of protein levels after vessel ligation and removal of the tissue. 24 Routinely, specimen samples obtained from biopsies or surgical procedures are examined by pathologists before further processing or formalin fixation.…”

“…Specifically, these guidelines do not mention phosphoepitopes that have been shown to be altered by delays in time-to-fixation. 18,20 There are relatively few studies or quantitative data about phosphoproteins and how their expression is affected by tissue extraction, preservation methods, processing, and time-to-fixation. The standard practices of ex vivo tissue handling are based on old protocols for tissue formalin fixation, often with no special care to reduce the time-tofixation.…”

Making personalized medicine a reality: the challenges of a modern translational research biopsy-driven program in an academic setting: the Segal Cancer Center experience

“…Thus, the deeper parts of the sample may not be properly fixed and may produce aberrant staining results when compared to core biopsies. These discrepancies apparently depend on the protein being analyzed, with phosphorylated epitopes such as phospho-AKT staining and phospho Erk1/2 being much more sensitive to time to fixation than the un-phosphorylated form of the proteins [10,11]. Indeed, this group recommends the collection of small biopsies even of resected surgical samples, so as to ensure a rapid and uniform formalin fixation.…”

Intrinsic indicators for specimen degradation

“…Therefore, the exact state of molecular degradation of individual human biospecimens is unknown. [4][5][6] The degradation stage of mammalian tissues after extended time of exposure to PAVs has been a central topic of forensic research. [7][8][9][10] Forensic research developed various methodologies to assess decay stage by measurement of chemicals, fatty acid, and PCR length.…”