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Last Updated: 06/04/15

Intrinsic and Extrinsic controls for Formalin Fixed, Paraffin Embedded (FFPE) tissue

Subcontractor: Yale University
Principal Investigator: David Rimm, M.D., Ph.D.
Co-Principal Investigator: David Hicks, M.D.

Dr. Rimm and his colleagues addressed the issue of tissue quality by performing and developing quantitative assessment of the effect of preanalytic cold ischemic time on protein expression in breast cancer tissues. Their team examined and quantified 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. The study was performed in collaboration with Dr. David Hicks from University of Rochester Medical center. Dr Hicks supplied 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. These sections were used to evaluate changes in antigenicity as a function of cold ischemic time.

Each antigen was measured using the AQUA method of quantitative immunofluorescence and was examined using a tissue quality index (TQI) model. The investigators 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, they 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. The team also noticed that phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in posttranslational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) were quite labile and loss of antigenicity was reported within 1-2 h of cold ischemic time.

Dr. Rimm and his colleagues concluded that key breast cancer biomarkers show no evidence of loss of antigenicity, although the dataset used, assessed the relatively short time beyond the 1-hour limit. Their team stressed the need for future studies that extend the time range and suggested the need for better normalization strategies to control tissue heterogeneity. The investigators also concluded that specimen collection should be closely monitored and subjected to quality control.

Vassilakopoulou et al., Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time, Lab Invest. 95(3), 2015; 334-41

Neumeister et al., A tissue quality index; an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue, Lab. Investigation, 94, 2014; 467-474

Neumeister et al., Quantitative Assessment of effect of Preanalytic cold ischemic time on Protein expression in Breast cancer tissues, J Natl Cancer Inst 2012:104; 1815-1824