ARTICLE |
Correspondence to: Hans-Anton Lehr, Institute of Pathology, Johannes Gutenberg University, Langenbeckstr. 1, 55101 Mainz, Germany.
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Summary |
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The benefit of quantifying estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer is well established. However, in routine breast cancer diagnosis, receptor expression is often quantified in arbitrary scores with high inter- and intraobserver variability. In this study we tested the validity of an image analysis system employing inexpensive, commercially available computer software on a personal computer. In a series of 28 invasive ductal breast cancers, immunohistochemical determinations of ER and PR were performed, along with biochemical analyses on fresh tumor homogenates, by the dextran-coated charcoal technique (DCC) and by enzyme immunoassay (EIA). From each immunohistochemical slide, three representative tumor fields (x20 objective) were captured and digitized with a Macintosh personal computer. Using the tools of Photoshop software, optical density plots of tumor cell nuclei were generated and, after background subtraction, were used as an index of immunostaining intensity. This immunostaining index showed a strong semilogarithmic correlation with biochemical receptor assessments of ER (DCC, r = 0.70, p<0.001; EIA, r = 0.76, p<0.001) and even better of PR (DCC, r = 0.86; p<0.01; EIA, r = 0.80, p<0.001). A strong linear correlation of ER and PR quantification was also seen between DCC and EIA techniques (ER, r = 0.62, p<0.001; PR, r = 0.92, p<0.001). This study demonstrates that a simple, inexpensive, commercially available software program can be accurately applied to the quantification of immunohistochemical hormone receptor studies. (J Histochem Cytochem 45:1559-1565, 1997)
Key Words: quantitative, immunohistochemistry, image analysis, estrogen receptor, progesterone receptor, personal computer
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Introduction |
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Evaluation of hormone receptor expression in tumor cell nuclei is an integral part of routine breast cancer diagnosis and provides important information with relevance for prognosis and choice of therapeutic approach. Today, the determination of estrogen receptor (ER) and progesterone receptor (PR) expression by immunocytochemistry is widely accepted (
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Materials and Methods |
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Immunocytochemical Studies
A total of 28 archival breast cancer cases were selected from a consecutive series of cases (infiltrating ductal carcinomas) submitted to the University of Washington for immunocytochemical studies of ER and PR expression. Information regarding the monoclonal antibodies (MAbs) used in this study, including clone designations, working dilutions, and sources, is summarized in Table 1. The anti-ER antibody and the anti-PR antibody used for the comparison with biochemical hormone receptor quantification were clone 1D5 and clone PR88, respectively. The comparison among three PR antibodies was performed using the antibodies designated by clone denomination as 1A6, 1A9, and mPRI (Table 1). MAbs to cytokeratin (CAM5.2) were included as a guideline for general tissue reactivity to antibodies and for quantitative estimation of the number of tumor cells present. Sections (4-5 µm) from representative blocks in each case were deparaffinized, rehydrated in graded alcohols, and subjected to heat-induced epitope retrieval using a microwave oven in 0.1 M citrate buffer (pH 6) for 8 min as previously described (
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Quantification of Immunocytochemical Staining
The technical setup included an Olympus BH-2 microscope (Olympus Optical; Tokyo, Japan), and a video camera (Javelin JE3462 RGB Video Camera) that transmits image data to a Macintosh 7500 computer, which comes equipped with 24-bit color digital imaging capabilities. All images were obtained using a x20 objective. The software used was Photoshop, version 3.0 (Adobe Systems; Mountain View, CA). Three x20 fields were chosen so as to best reflect the overall immunostaining of the tumor contained on the entire slide. For the entire study, the camera had its auto mode turned off and manual controls were used to adjust the image intensity, which was kept at an identical level during the entire study. For longitudinal studies over longer time periods, standardized control slides should be used every day, with proper calibration of the microscope and camera to yield comparable conditions for image collection. The digitized images were stored on an external data storage device (ZIP drive; Iomega; Roy, UT). The procedure for determination of immunostaining intensity is shown in Figure 1. Using the Magic Wand tool in the Select menu of Photoshop, the cursor was placed on an ER/PR-positive nucleus. The tolerance level of the Magic Wand tool was adjusted so that the entire nucleus was selected. Using the Similar command in the Select menu, all immunostained nuclei were automatically selected. Subsequently, the image was transformed to 8-bit grayscale. An optical density plot of the selected are was generated using the Histogram tool in the Image menu. The mean staining intensity (in arbitrary units, AU) was recorded. Subsequently, the background was selected using the Inverse tool in the Select menu, and immunostaining was quantified using the Histogram tool in the Image menu. Immunostaining intensity was calculated as the difference between nuclear immunostaining and background immunostaining and was designated immunocytochemical index with arbitrary units (AU). A representative example of an immunocytochemical study and its selection of nuclear area and background is shown in Figure 2.
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Determination of Hormone Receptor Expression by DCC and EIA
Two independent methods (DCC, EIA) of quantification of ER and PR on the same tumor cytosol preparations were employed (
Statistical Analysis
For both ER and PR, quantitative immunostaining results (immunocytochemical index) were compared to the DCC and EIA assays of receptor content. Because the dilution studies suggested a possible linear log relationship (not shown) between the quantity of antibody deposited on the specimen and the immunocytochemical index, the quantitative immunostaining results were plotted against the log of the biochemical and immune assay results. This interrelation is also analogous to previous studies (
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Results |
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Immunocytochemical Staining
Most breast carcinomas demonstrated a heterogeneous immunostaining pattern for both ER and PR in different areas of the tumor. Background staining was uniformly low, and no cytoplasmic or membranous immunoreactivity pattern was observed. The quality of fixation was monitored by immunostaining with an antibody to cytokeratin 8 (CAM 5.2), which is routinely used in our laboratory to identify improperly fixed tissues and to select fields for interpretation of the immunocytochemical receptor studies. Those areas were selected for image analysis, in which the immunostaining intensity allowed unequivocal differentiation between nuclei and background (Figure 2). Determination of nuclear and background staining intensity was possible in all of the representatively selected cases.
Correlation Between Hormone Receptor Expression by Immunocytochemical and Biochemical Techniques
The scatterplots demonstrating the correlations between immunocytochemical and biochemical techniques for the quantification of ER and PR are shown in Figure 3 and Figure 4, respectively. The immunostaining intensity ranged from 0 arbitrary units (AU) (weak immunoreactivity) to 135 AU (strong immunoreactivity) for ER and from 0 AU (weak immunoreactivity) to 149 AU (strong immunoreactivity) for PR. The ER values by the DCC technique and EIA were from 2-695 fmol/mg and from 0-444 fmol/mg, respectively. The PR values by DCC and EIA were from 2-292 fmol/mg and 1-568 fmol/mg, respectively. Analogous to previous studies (
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To demonstrate the clinical applicability of the Photoshop-based image analysis, we also compared, in 12 breast cancer cases, the quantitative immunostaining of three different antibodies to different epitopes of the PR molecule. When these were quantified by Photoshop image analysis, we found that one of the antibodies (1A6) yielded a significantly weaker immunostaining intensity than the other two antibodies (Figure 5).
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Discussion |
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The principal finding of this study is the demonstration that a simple, inexpensive, and commercially available software program (Adobe Photoshop) can be applied to the quantification of immunocytochemical hormone receptor studies. This application in routine diagnostic pathology follows up a recent report in which we have applied Photoshop-based image analysis with custom-made plug-ins for the quantification of Ki-67-defined proliferative activity in breast cancer (
We have correlated the hormone receptor data obtained by Photoshop with two different techniques of biochemical receptor quantification. The DCC technique, which uses fresh-frozen tumor material, was chosen because of its historical role in hormone receptor diagnosis and because most previous immunocytochemical studies have compared their results with this "gold standard" in hormone receptor quantification. The EIA was chosen because it allows comparison of an immune-based assay with immune-based tissue staining. Furthermore, the immune-based assay overcomes some of the potential limitations of the DCC technique, including the potential for endogenous estrogens to interfere with radioligand binding (
The Photoshop-based image analysis yielded qualitative data that showed a statistically significant correlation with both the DCC technique and the EIA (Figure 3 and Figure 4). Indeed, the correlations for ER and PR are similar to those previously reported for diverse image analysis systems (
The benefit of quantifying hormone receptor expression beyond the determination of negative vs. positive has been well established (
We believe that the availability of a simple inexpensive image analysis system for use in routine pathological diagnosis of breast cancer cases makes it possible to provide a large database to further study the impact of hormone receptor expression on prognosis and/or response to therapy and on other questions related to hormone receptor expression. In a next step, we will be using this technique to compare hormone receptor immunostaining with hormone receptor visualization and quantification via positron emission tomography (PET) (
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Acknowledgments |
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Supported in part by NIH grants CA-36250 and CA-42045.
We thank Phyllis Davie, Liz Donato, Janice Morihara, Tracie Evans, Farinaz Shokri, and Raquel Dobbins for their outstanding help with the immunocytochemical studies.
Received for publication January 24, 1997; accepted June 9, 1997.
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