Abstract P1-07-05: Evaluation of tissue processing factors affecting HER2 IHC staining intensity in breast cancer cell lines
Authors: Jensen K , Erickson J , Webster S , Pedersen HC 
Published: August 2014

Abstract
Background: In breast cancer the HER2 status as established by IHC (e.g. HercepTest™) determines eligibility for HER2 targeted therapy.
Pre-analytical procedures including tissue processing create variation in HER2 intensity seen in breast cancer tissues. Therefore, there is a need for a greater understanding of the effects of the individual tissue processing steps on HER2 staining intensity.
Control cell lines (CCL) are commonly used as staining run controls in immunohistochemistry (IHC) in both pharmacodiagnostic, routine and in clinical research. As a surrogate for cancer tissue, these immortalized cancer cells can serve as a model system for investigating the effect of process variables on staining intensity. This study was designed to investigate if variations in cell/tissue processing (fixation, embedding and sectioning) affect the IHC staining intensity in a HER2 formalin- fixed, paraffin-embedded (FFPE) control cell lines.
To introduce a more objective scoring we have evaluated the application of digital image analysis combined with design of experiment (DOE) statistics.
Methods: The following tissue processing parameters were investigated: embedding medium (HistoGel™ or agarose), fixation time (18 or 48 hours in neutral buffered formalin), density of cells (25% or 50%) and thickness of cut sections (2 μm, 4 μm or 6 μm). Factors were set up in a 2k factorial experimental design; all slides were stained with Dako HercepTest™. All slides were scanned, analyzed and compared on two different image analysis systems: ACIS®III (Dako) and Scanscope CS (Aperio).
The DOE statistical analysis revealed the contribution of individual factors as well as any additive effect of a combination of factors. Analysis was based on the level of intensity as well as histoscores.
Results: In a 24 DOE experiment, several of the parameters described above were associated with decreased staining intensity: increased fixation time (p &lt; 0.001), decreased section thickness (p &lt; 0.001) and decreased cell density (p &lt; 0.001). In a separate 23 experiment, it was shown that fixation media in combination with cell density was associated with changes in staining intensity (p values from 0.001 to 0.022). Three different algorithms and two different slide scanners were tested and results were consistent regardless of the image analysis system or the algorithm used.
Conclusions: This study identified several tissue processing factors which impact IHC staining intensity in a HER2 expressing CCL. The discovery of these effects has advanced the understanding of processing methodology and the influence of pre-analytical factors on HER2 IHC staining intensity.
The application of a DOE setup with quantification by image analysis can be a useful tool for the systematic evaluation of several variables compared with the standard “one factor at a time” design. A multifactor effect between fixation media and cell density was detected which would not have been possible in a “one factor at a time” design. Implementing the knowledge achieved from this study provides a methodology for standardizing tissue processing and thereby improving HER2 testing in breast cancer.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-05.