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

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.

Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues.

BACKGROUND: Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. METHODS: A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. RESULTS: We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. CONCLUSIONS: Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.

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

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, with 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. Epitopes phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, estrogen receptor (ER), Ki67, cytokeratin and GAPDH were assessed. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared with biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, ER and cytokeratin showed significant loss of antigenicity. This data suggest 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.