Affiliation of authors: Department of Pathology, Yale University School of Medicine, New Haven, CT
Correspondence to: David L. Rimm, MD, PhD, Department of Pathology, Yale University School of Medicine. 310 Cedar St., P.O. Box 208023, New Haven, CT 06520-8023 (e-mail: david.rimm{at}yale.edu).
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ABSTRACT |
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INTRODUCTION |
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One method to reduce the number of variables in immunohistochemistry analysis is the use of tissue microarrays. Many laboratories have used this technology to study the expression of biomarkers in hundreds of tumor samples on the same slide (4,5). Comparisons between tissue microarrays containing breast cancer samples and corresponding whole-tissue sections have demonstrated that, for some tissues, only a single tissue microarray core (i.e., a histospot) is sufficient to identify antigen expression in the whole section when the heterogeneity is averaged across a whole population of tumor samples, with high concordance for common biomarkers and reproducible prognostic associations between staining levels and clinical outcomes (6,7). This approach eliminates differential antigen retrieval and staining conditions as possible variables. However, it does not eliminate the variability in scoring. The current standard methods for scoring protein expression by immunochemistry of traditional tissue sections or tissue microarrays is the 03 scale (in which 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) or the H score (a product of staining intensity and the percent of cells stainedfor example, if 50% of the slides shows an intensity of more than 3, 20% show an intensity of more than 2, and 30% are negative, then the H score would be 150 + 40 = 190). These scoring methods are subjective and are subject to human variability even within controlled settings, i.e., in which positive and negative controls are used (8).
We have recently developed an automated scoring system for assessing biomarker expression in tissue sections called the automated quantitative analysis (AQUA) system (9). The AQUA system is linked to a fluorescent microscope system that detects the expression of biomarker proteins by measuring the intensity of antibody-conjugated fluorophores within a specified subcellular compartment (typically including the nucleus, cytoplasm, and plasma membrane) within the tumor region of each tissue microarray spot. The result is a quantitative score of immunofluorescence intensity for the tumor. An AQUA analysis removes the subjectivity of the traditional scoring system and provides more continuous and reproducible scoring of protein expression scoring in tissue samples (9).
In this study, we use the AQUA system to measure the expression of HER2, estrogen receptor (ER), and p53 proteins in breast cancer tumors and to measure the expression of HER2 in a series of control cell lines to determine its absolute expression in each cell line. Our goal was to use the AQUA system to investigate how the antibody concentration used in immunohistochemical staining affects the association between apparent protein expression in the tumor and patient outcome. We also used a range of HER2 antibody concentrations on a series of cell lines that express HER2 to assess the dynamic range of HER2 protein expression (i.e., the range of HER2 concentrations in cells from those with low HER2 expression to those with high HER2 [i.e., HER2 amplified] expression) in a population of tumors.
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PATIENTS AND METHODS |
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Tissue microarrays were constructed from tumor tissue core samples. Core samples were obtained from representative regions of sections from each tumor that had been selected by use of the corresponding full sections stained with hematoxylin and eosin. The tissue microarray was constructed with single core samples (0.6-mm diameter) from each tumor that were spaced 0.8 mm apart in a grid format by using a tissue microarrayer (Beecher Instruments, Silver Spring, MD), as previously described (6,7). The tissue microarray was cut into 5-µm sections with a microtome, and the sections were adhered to the slide by means of an adhesive tape-transfer method, as described by the manufacturer (Instrumedics, Inc., Hackensack, NJ), and cross-linked to the slide with UV irradiation according to manufacturer's instructions.
Patient Cohort Characteristics
The patient cohort consisted of a total of 250 patients with invasive breast carcinoma (125 with lymph nodenegative disease and 125 with lymph nodepositive disease) with tumor tissue available in formalin-fixed paraffin-embedded tissue blocks. These 250 patient samples were selected at random and on the basis of adequate tumor availability from a collection of more than 700 patient samples obtained from archives at the Yale University Department of Pathology; the original tumors had been resected between January 1, 1962, and December 31, 1977, and attempts were made to collect every tumor sample during that period. However, because of missing or exhausted tissue blocks, some samples could not be retrieved and included in this study. The follow-up time ranged from 2.4 months to 41.5 years (median = 8.3 years), and age at diagnosis ranged from 24 years to 86 years (median = 59 years). More detailed information about this cohort is published elsewhere (10,11). All personal health information was collected under the approval of Yale University Human Investigation Protocol 8219, which approved the informed consent signed at the time of surgery.
Tissue Microarray Immunohistochemical Staining
The tissue microarray slides were deparaffinized by two xylene rinses followed by two rinses with 100% ethanol. Antigen retrieval was performed by boiling the slides in a pressure cooker filled with 7.5 mM sodium citrate (pH 6.0). After rinsing briefly in 1x Tris-buffered saline (TBS) at pH 8, slides were incubated for 30 minutes in 2.5% hydrogen peroxide in methanol to block endogenous peroxidase activity. Slides were then incubated with 0.3% bovine serum albumin in 1x TBS for 1 hour at room temperature to reduce nonspecific background staining and then subjected to washes in 1x TBS, in 1x TBS containing 0.01% Triton, and then in 1x TBS, each 2 minutes long (hereafter referred to as TBS rinses). Slides were incubated first with a mouse anti-cytokeratin monoclonal antibody (clone AE1/AE3, DAKO, Carpinteria, CA; diluted 1 : 200) for the HER2 slides or with a rabbit anti-cytokeratin polyclonal antibody (Zymed, South San Francisco, CA; diluted 1 : 50) for the ER and p53 slides overnight at 4 °C to define the epithelial mask. Slides were rinsed in 1x TBS and then incubated with HER2 antibody (c-erbB-2 oncoprotein, DAKO; diluted 1 : 500 through 1 : 8000), ER antibody (ER- mouse anti-human clone ID5, DAKO; diluted 1 : 100 or 1 : 1000), or p53 antibody (mouse anti-human clone DO-7, DAKO; diluted 1 : 50 through 1 : 800) for 1 hour at room temperature. Slides were rinsed in TBS as described above and incubated with secondary antibodies for 1 hour at room temperature: biotin anti-mouse or biotin goat anti-rabbit secondary antibodies (Vector Laboratories, Burlingame, CA; diluted 1 : 200) or mouse or rabbit secondary antibodies attached to a dextran-polymer backbone that was decorated with more than 100 molecules of covalently attached horseradish peroxidase (called "Envision," DAKO) for HER2, ER, and p53 studies. The slides were washed with the TBS rinses described above and incubated for 30 minutes at room temperature with Alexa 546-streptavidin (Molecular Probes, Eugene, OR; diluted 1 : 200) to label the cytokeratin and then for 10 minutes with Cy-5 tyramide (NEN Life Science Products, Boston, MA) to allow coupling of Cy-5 dyes adjacent to the horseradish peroxidaseconjugated secondary antibody (tyramide is activated by horseradish peroxidase, and the activated form interacts covalently with adjacent protein molecules) for HER2, ER, and p53 studies. The emission peak of Cy-5 falls outside the tissue autofluorescence spectrum, thus minimizing background fluorescence for more accurate quantification of signal. The slides were then stained with the DNA staining dye 4',6-diamidino-2-phenylindole (DAPI) for 10 minutes, mounted with an antifade medium containing 0.6% n-propyl gallate in glycerol, and covered with a cover slip.
For the diaminobenzidine-based brown staining for HER2 expression, slides were prepared as described for the primary antibody incubations and washes, and then slides were incubated with Envision (DAKO) for 1 hour at room temperature. This incubation was followed by TBS rinses, visualization with diaminobenzidine (diaminobenzidine chromogen, DAKO), and then counterstaining with ammonium hydroxideacidified hematoxylin. The slides were mounted with ImmunoMount (Shandon, Pittsburgh, PA) and then analyzed by use of a conventional four-point scoring system for HER2 membrane expression (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining). Slides were read by two observers who were blinded to the outcome data for each slide. A high concordance (>90% exact agreement) was found between their scoring.
Cell Lines
The cell lines SK-BR-3, BT-474, MDA-MB-453, MDA-MB-435S, BT-549, T-47D, SW-480, MCF-7 MDA-MB-231, and MDA-MB-361 were purchased from the American Type Culture Collection (Manassas, VA), and all are breast cancer lines except for SW-480 cells, which are a colon cancer line. BAF3 cells, an interleukin 3dependent cell line, were obtained from a laboratory in the Department of Genetics at Yale University. SK-BR-3, BT-474, SW-480, MCF-7, MDA-MB-453, and MDA-MB-435S cells were routinely cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and 1% penicillinstreptomycin (Life Technologies, Inc., Grand Island, NY). T-47D and MDA-MB- 231 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillinstreptomycin (Life Technologies). BAF3 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillinstreptomycin, and 10% WEHI-cell conditioned medium (12), as a source of interleukin 3. All cell lines were maintained in a 37 °C incubator with 5% CO295% air (Life Technologies).
Cell Line Immunofluorescent Staining for HER2
SK-BR-3, BT-474, MDA-MB-361, and MDA-MB-453 cells were trypsinized and plated at intermediate cell density into six-well plates (Life Technologies), grown overnight, and then fixed with neutral-buffered 10% formalin. BT-549, T-47D, SW-480, MCF-7, MDA-MB-231, BAF3, and MDA-MB-435S cells were trypsinized and then fixed in Shandon cytospin collection fluid (Thermo Electron, Pittsburgh, PA). Cells were stained as described above for the tissue microarray sections for HER2.
HER2 Detection by Enzyme-Linked Immunosorbent Assay
To measure the concentration of HER2 protein in cell lines, HER2 protein was detected with the DuoSet IC Human Total ErbB2 enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN), in which lysate production and the assay were as described by the manufacturer's protocol. HER2 concentrations were calculated from the resultant standard curve and are expressed as picograms per microgram of total cell lysate. All standards and samples were measured in duplicate.
Evaluation of Staining by the AQUA System
The AQUA system was used for automated image acquisition and analysis as previously described (9). In brief, images of the tissue microarray core sections (histospots) and cell lines were captured with an Olympus BX51 microscope and analyzed with the AQUA software. For each histospot, areas of tumor are distinguished from stromal elements by creating an epithelial tumor mask from the antikeratin protein signal, which was visualized via the Alexa 546 fluorophore. The tumor mask was determined by gating the pixels in this image, in which an intensity threshold was set by visual inspection of histospots, and each pixel was recorded as "on" (tumor) or "off" (nontumor) by the software on the basis of the threshold. The DAPI image, which was used to identify the nuclei, was subjected to a rapid exponential subtraction algorithm that improves signal-to-noise ratio by subtracting the out-of-focus image from the in-focus image. After application of the rapid exponential subtraction algorithm, the signal intensity of the target antigen (e.g., HER2, p53, or ER), which was acquired under the Cy5 signal, was scored on a scale of 0255. The AQUA score within the subcellular compartments (i.e., nucleus and membrane) was calculated by dividing the signal intensity by the area of the specified compartment. The AQUA score for the cell lines was determined by dividing the signal intensity by the total area under the tumor mask.
Statistical Analysis
Statistical analyses for HER2 and p53 protein expression in the tumor microarrays were completed with X-tile (13) and StatView version 5.0.1 (SAS Institute, Inc., Cary, NC). Survival was calculated by the KaplanMeier method, and statistical significance of the association between biomarker expression and survival was determined by the MantelCox log-rank test. Plots of univariate relative risks (RRs) of mortality as a function of cutpoint of biomarker expression and antibody concentration were constructed by X-tile. All statistical tests were two-sided.
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RESULTS |
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To assess the relationship of AQUA score, biomarker protein concentration, and antibody concentration in cell lines, we used HER2 because its expression has been measured in many cell lines (16,17). Published data (16,17) indicate that an effective assay for HER2 protein must be able to detect from less than 103 HER2 molecules per cell to greater than 106 HER2 molecules per cell. However, because levels of HER2 expression vary among cell lines, we selected a series of cell lines (Table 1), measured HER2 protein levels in these cell lines by ELISA, and compared our results with previously published gene amplification data (1821). One group of cell lines had high HER2 protein expression and multiple copies of the HER2 gene (i.e., B-T-474, SK-Br-3, MDA-MB-361, and MDA-MB-453), and the other group of cell lines lacked or had low HER2 protein expression and no detectable HER2 gene amplification (i.e., T-47D, MDA-MB-435S, BT-549, SW-480, MCF-7, MDA-MB-231, and BAF3). The results of our ELISAs for the construction of the standard curves were more or less consistent with published values (16,17).
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DISCUSSION |
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The traditional "brown stain" immunohistochemistry analysis, although robust and well accepted, is designed to detect context (i.e., tissue regions that are stained are compared with those regions that are not stained) rather than to linearly measure intensity across a broad dynamic range. The use of the AQUA assay expands the range of immunoassays and allows better quantification. However, both conventional brown stainbased and immunofluorescence-based assays have a limited concentration range because of the enzyme amplification used and other factors. We estimate the functional range for conventional brown stainbased assays is between 1 and 1.5 orders of magnitude (data not shown). The functional range for fluorescence-based assays is between 1.5 and 2.5 orders of magnitude (see Fig. 4). This limitation may decrease the accuracy of the analysis and also obscure associations with expression because of the saturation or insufficient sensitivity of the assay. This problem may explain some of the variability in the p53 and HER2 literature and may also explain the U-shaped relationship that has been previously observed between HER2 expression and outcome (13,14). However, this study has limitations. Specifically, the number of patients in each study set is relatively small, and thus the results should be validated in both larger cohorts and with other antibodyantigen pairs.
In summary, this study highlights the problems of subjective analysis of immunohistochemistry studies. When a high concentration of antibody was used, subtle differences in the level of HER2 expression in cells with low-level HER2 expression could be discerned, but because of assay saturation, cells with high-level HER2 expression could not be measured. Conversely, when a low concentration of antibody was used, HER2 levels in cells with a high-level HER2 expression could be distinguished from those in cells with very high-level HER2 expression; however, this concentration of antibody could not distinguish HER2 levels in cells with low-level HER2 expression from background. A similar relationship was observed for p53 and may also be apply to other biomarkers, although not to ER. The nonquantitative nature of conventional immunohistochemistry studies and the lack of validated control cell lines that can be used to make standard curves for biomarker expression levels can result in incomplete or even conflicting conclusions.
The implications of our results for current practice are unclear. This study illustrates that a greater awareness of an antigen's dynamic concentration range is needed to rigorously assess tissue biomarker associations with outcomes. However, it is not clear that conventional brown stain immunohistochemistry can be used for this task. As the requirement for increased accuracy for measurement of in situ protein concentration increases, new methods, such as the AQUA system, may be used more often. We believe it is likely that exact amounts of multiple proteins in specific subcellular compartments will be measured to assist in the biological characterization of a tumor, such as identifying pathways that are activated or inactivated in cancer, to aid in the selection of targeted therapies. We will probably see the increased use of validated control cell lines to ensure that the appropriate concentration of antibody can be used to detect the antigen of interest in the linear range of the assay. This level of accuracy, quality control, and quality assurance has been established in many areas of evidence-based medicine, so that we believe that it should be only a matter of time before anatomic pathology establishes the same standards for its assays.
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NOTES |
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A. McCabe and M. Dolled-Filhart contributed equally to this work.
Marisa Dolled-Filhart is supported by the United States Army Breast Cancer Research Grant DAMD17-03-1-0349. Dr. Camp is supported by NIH Grant K0-8 ES11571, and both Drs. Rimm and Camp are supported by the Breast Cancer Alliance of Greenwich, CT. Dr. Rimm is supported by a grant from the Patrick and Catherine Weldon Donaghue Foundation for Medical Research, NIH grant NCI R21 CA100825, and United States Army Grant DAMD-17-02-0463.
Funding to pay the Open Access publication charges for this article was provided by the Department of Pathology, Yale University School of Medicine.
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Manuscript received March 24, 2005; revised October 7, 2005; accepted October 28, 2005.
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