A novel approach to automated quantitative analysis of HER2 expression
Authors: Holden Elena, Ed Luther
Journal: Clinical Cancer Research (2008)

AACR Centennial Conference: Translational Cancer Medicine-- July 20-23, 2008; Monterey, CA

B17 

Objectives     In the emerging paradigm of personalized medicine, accurate quantification of cellular expression of relevant proteins linked to particular therapies is of vital importance. Performance requirements (accuracy, sensitivity, precision) for quantitative tissue-based tests continue to rise.    In this study we establish the following goals:    a. Develop a novel approach to tissue analysis by utilizing an unbiased automated method of scanning and analysis: continuous “scoring” instead of the traditional “0” to “3+“ approach.    b. Compare data sets derived from fluorescently and chromatically stained samples. Fluorescence labeling is perceived to be a more quantitative approach whereas chromatic staining is the mainstream pathology approach.    c. Evaluate standardization and quality control materials for automated HER2 expression analysis.    Methods and results     Analysis was performed on an iCys® Imaging Cytometer (CompuCyte, MA). The process was fast and objective, requiring a minimal degree of technician intervention with the supporting image data archived in an accessible format. Chromatically and fluorescently stained samples were analyzed. To validate the accuracy of quantification of HER2 analysis, cell block tissue microarrays were constructed from cell lines with known levels of expression of HER2. Serial sections of the arrays were stained with HER2HercepTest IHC (chromatic staining) and fluorochromes Alexa Fluor 488 and Alexa Fluor 647. The mean values of HER2 staining intensity were obtained for each of the core elements as continuous quantitative data. The results obtained from fluorescent detection methods (Alexa Fluor 488/647) were similar to those obtained from the chromogenic (DAB) method. Automated analysis results were in close agreement with those obtained by manual scoring by a pathologist.    Novel standardized peptides that mimic HER2 native antibody binding regions were used as staining controls and applied to slides as duplicate spots at four different concentrations. Cell block cores of cell lines with assigned expression levels of HER2 were applied to the peptide slides. Serial slides were stained with antibodies to HER2, at different concentrations and developed with DAB. Analysis of the peptide spots showed a correlation between the measured absorbance levels and the peptide concentrations, allowing generation of a calibration curve relating the measured laser light absorbance to the HER2 peptide concentration. Linear correlation was established between laser light absorption measurements and known HER2 expression levels of the cell lines. Combining both sets of data produced a calibration curve relating the antibody standard concentration to HER2 expression levels of reference cell lines, opening the opportunity for accurate quantification of HER2 expression levels in patient specimens.    Conclusions     Our study provides evidence that HER2 expression can be accurately and reproducibly quantified by automated imaging cytometry, using both fluorescent and chromatic dyes to measure a continuum of protein expression levels. It offers a higher degree of “resolution” compared to currently employed manual or operator-assisted semi-automated microscopy methods. Standardized peptides evaluated in this study hold high promise as future standards for the development of accurate quantitative HER2 expression analysis and as quality control material.