Abstract 1141: Quantitating signaling pathway proteins in FFPE sections using multispectral imaging and automated image analysis
Authors: Hoyt Clifford , Lane Kristin , Gardner Humphrey , Feldman Michael 
Published: February 2011

Abstract
Background: A common goal in pharmaceutical research is to reveal correlations between signaling pathway activity in intact tissue and clinical outcomes. Correlations support target validation, trial design, patient selection, response assessment, and, if trials are successful, the diagnostic component of theranostics. However, the predictive power of measurements of protein expression depends on sample preparation and the tools used to quantitate label ‘intensity’. In particular, ischemic time and rate of fixation are known issues for labile proteins, and, in the case of immuno-fluorescence (IF), signals can be obscured by autofluorescence, thus increasing apparent signal.
Objective: The goal of this work is to evaluate some practical approaches to increasing the reliability of quantitative IHC and IF on formalin-fixed, paraffin-embedded samples. A first set of tests compare protein preservation with needle biopsy and resection, expecting that the reduced ischemic time and favorable formalin penetration rate for biopsies benefits preservation. A second set of tests evaluates the benefit of spectral imaging for accurately quantitating multiplexed IF labeling.
Methods and Materials: Biopsy vs. resection tests used single-color IHC (DAB with hematoxylin counterstain) applied to serial sections, to assess relative protein expressions of six commonly studied signaling pathway proteins: pAKT, pEGFR, pERK, pS6, pSTAT3, and pVEGFR. For each slide, three 20x multispectral images were acquired of representative tumor tissue. IHC signals were spectrally unmixed and isolated counterstain. Machine-learning-based automated image analysis was used to differentiate relevant tissue regions (e.g., malignant and normal epithelia, stroma, necrosis, etc.) and segment cellular compartments (nuclei, cytoplasm, and membrane) to allow for detailed, spatially resolved IHC quantitation. Stain optical density, as a quantitative measure of stain ‘intensity’, on a per-cell basis was analyzed to determine average protein expression. The benefits of spectral imaging for IF labeling were evaluated using a cocktail for pAKT, pERK, and pS6, applied to a test tissue micro array. Signals of each label were measured using conventional fixed-filter approaches and using spectral imaging and associated unmixing algorithms.
Results and Discussion: Data shows a clear advantage to using biopsy material versus resection material, with some phospho-proteins showing almost complete loss in resection material. Spectral unmixing demonstrated that conventional filter methods markedly overestimate individual channel signals, as they fail to account for background and cross talk, severely limiting the dynamic range of pathway activation studies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1141.