P1-07-03: Preanalytical Variables Affect Protein Expression in Formalin Fixed Paraffin Embedded Tissue – Assessment of Intrinsic Controls To Define Tissue Quality for Immunohistochemical Analysis.
Authors: Neumeister VM , Lostritto K , Siddiqui S , Anagnostou V , Vassilakopoulou M , Zarrella EA , Molinaro AM , Hicks DG , Rimm DL 
Published: June 2012

Background: Recently it has been shown that biospecimen handling and pre-analytical variables can dramatically affect biomarker assays of protein expression in tumor tissue. Phospho-proteins and even labile unmodified proteins have been suggested to show significant loss of expression due to prolonged time to formalin fixation. Here we assess 4 clinically relevant proteins (ER, PR, HER2 and Ki67) and 20 other proteins for changes as a function to the key preanalytic variables of ischemic time. The ultimate goal of our effort is to find a method to monitor the degradative effect of these variables by construction of a Tissue Quality Index (TQI).

Materials and Methods: Two different breast cancer cohorts were used in order to analyze the biomarkers and their change according to time to formalin fixation. The first cohort consists of 93 breast cancer specimens in 2 fold redundancy on a TMA with cell lines and controls. The time to formalin fixation for each breast cancer specimen was recorded and ranges from 25 to 415 minutes. The second cohort consists of 25 matched pairs of breast cancer biopsies and resections. The time to formalin fixation for the biopsies is minimal while the time to fixation for the resections, though not recorded, averages between 1 and 3 hours. Protein expression was measured using the AQUA method of quantitative immunofluorescence.

Results: ER alpha, PR, HER2 and Ki67 were each analyzed on the time to fixation array with 2 different antibodies commonly used in the clinical setting. Correlation of AQUA scores of these markers with time to formalin fixation revealed a trend towards loss of protein expression as a linear function of time to fixation without reaching statistical significance. Analysis of these 4 proteins on the matched pairs of biopsies and resections showed that tumor heterogeneity predominated over the effects of ischemic time. Toward identification of markers for a TQI, 20 biomarkers were analyzed on the time to fixation array. Both HIF1alpha and AKAP13 show a significant increase as a function of time to fixation, whereas pMAPK, histone 4 and pTyrosine 4G10 revealed a significant loss of expression. These trends were confirmed in the matched pair validation set with the execption of histone 4. A TQI is being built from these variables.

Conclusions: Ischemic time is a critical pre-analytical variable that impacts measurement of protein expression in tumor tissue. The 4 standard markers used clinically in breast cancer appear to show only moderate effects that appear less critical to measurement accuracy than the issue of tumor heterogeneity. We identified 4 proteins which show a significant change with increasing time to formalin fixation and should allow construction of a TQI for assessment of pre-analytic antigenic degradation.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-03.