For the last 30 years, determination of estrogen receptor (ER) and progesterone receptor (PR) expression in breast carcinomas has played an important role in the management of breast cancer because these biomarkers are highly predictive for a clinical benefit from targeted endocrine therapy.1–3 The clinical importance of ER, in particular, in breast cancer management has made its assessment mandatory for every newly diagnosed patient,4 and thereby a successful example of the use of biomarkers in guiding targeted cancer therapy.
In current clinical practice, the analysis of ER and PR by immunohistochemistry (IHC) is an important part of the initial pathologic evaluation of breast cancer,5,6 and the recent inclusion of these biomarkers in the clinicopathologic surrogate definition of intrinsic breast cancer subtypes by the St. Gallen expert consensus7 emphasizes the importance of standardization of preanalytical, analytical, as well as postanalytical variables in order to help ensure the reliability, quality, and accuracy of the results.8 Evidence-based consensus guidelines from the joint recommendations of the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) have attempted to address the important issue of assay standardization, and have stressed the importance of quality control and quality assurance programs for laboratories performing ER and PR assessment.6
In recent years, improvements have been made in IHC methodology with the introduction of polymer-based detection,9 the development of highly sensitive and specific monoclonal antibody reagents,10,11 and the introduction of new automated test platforms that provide unparalleled accuracy, efficiency, and reproducibility.12 With the introduction of new test platforms and new IHC regents for ER and PR analysis, there is a need for rigorous evaluation and technical validation of their performance before their clinical implementation.
Here, we describe a series of multicenter studies that examined the performance characteristics of rabbit monoclonal anti-ER (clone EP1) antibody and mouse monoclonal anti-PR (clone PgR 1294) antibody for a new automated staining platform (Dako Omnis). These antibodies are in development and are not currently commercially available.
MATERIALS AND METHODS
The specimens were residual tissue specimens from individual breast cancer patients whose identities were not traceable. Tissue blocks were obtained from Dako (Carpinteria, CA and Glostrup, Denmark) and from a commercial source (Cureline, South San Francisco, CA) who in turn collected them from at least 15 different hospitals or clinical sites. All tissues were formalin-fixed and paraffin-embedded and the majority were fixed for 6 to 72 hours according to ASCO/CAP fixation guidelines. Study activities were performed in accordance with the ethical principles that have their origin in the Declaration of Helsinki and Good Clinical Practice. Approval of the study protocols by ethics committees was not required as no human subjects were directly involved. Quality control was performed on the specimens to confirm a diagnosis of invasive breast cancer, and the specimens were prescreened to determine the extent of nuclear ER or PR expression (see Table 1 for antibodies used for prescreening).
Antibodies and Reagents
The antibodies used for these studies are shown in Table 1. The antibodies were configured as FLEX Ready-to-Use and used with the EnVision FLEX, High pH visualization system according to the manufacturer’s instructions for use. At each testing laboratory, reagents from 1 lot of reagents was used for staining on 1 Dako Omnis automated staining platform. All antibodies and associated reagents were obtained from Dako (Glostrup, Denmark).
Overall Study Design, Stratification of Specimens, and Observer Blinding
Slides with tissue sections (4 μm thick) were prepared from each specimen at Dako and labeled with the study number, and a unique randomization number that was not related to the specimen ID. The slides were distributed to each testing laboratory for staining in a prespecified randomized order according to the protocol for each study. Also provided were prestained hematoxylin and eosin sections from each specimen, and unstained tissue control slides with positive control (cervix) and negative control (colon epithelium) tissues. The tissue controls were fixed in the same manner as the test tissues.
The method comparison studies tested the concordance (agreement) between automated staining on the Dako Omnis platform with either the anti-ER clone EP1 or anti-PR clone PgR 1294 and staining on the Autostainer Link 48 instrument (clone EP1 for ER; clone PgR 636 for PR). Tissue specimens were stratified into 2 groups of 125 specimens for the ER study and 3 groups of 100 specimens for the PR study in order to ensure an even distribution of specimens to the various testing laboratories (UCL, IEO, URMC) with regards to ER or PR expression.
The reproducibility studies tested the interrun (day-to-day), interlaboratory, and interobserver reproducibility of automated staining on the Dako Omnis platform with either the anti-ER clone EP1 or anti-PR clone PgR 1294. The studies were conducted at 3 laboratories; each performed 5 staining runs on 20 specimens on 5 nonconsecutive days over a period of at least 20 days. At each laboratory, 1 trained pathologist evaluated the sections stained at that laboratory. When the scoring was complete, the stained slides from laboratories 1 (UCL) and 2 (IEO) were forwarded to laboratory 3 (URMC) where they were evaluated by 3 independent pathologists.
Deparaffinization and antigen retrieval were done using EnVision FLEX High pH Target Retrieval Solution for Autostainer Link 48 or Dako Omnis. Control tissues and universal negative control antibody reagents (Table 1) were processed in parallel with tissues exposed to the primary antibodies. Stained tissue sections were dehydrated and coverslipped following routine procedures. The slides, which were blinded for specimen ID and staining method, were evaluated by a pathologist in the randomized order using a bright field microscope. Specimens were evaluated for several parameters related to staining quality, including artifacts and levels of background cytoplasmic staining in tumor and nontumor cells. Nuclear ER or PR staining was scored using the ASCO/CAP guideline,6 which defines a positive result as nuclear staining of ER or PR in ≥1% of invasive breast carcinoma tumor cells. For the method comparison studies, nuclear ER or PR staining was also recorded for other cell types if present, including ductal carcinoma in situ (DCIS) and nontumor cells.
Data Collection and Statistical Analysis
Study data were entered directly into an online electronic data capture system (Viedoc; Pharma Consulting Group, Uppsala, Sweden). Once the last data were entered and the study databases were locked, the data were unblinded for specimen ID using the randomization key for each study. Two-sided Wilson Score 95% confidence intervals were calculated for the study endpoints using JMP software (SAS Institute, Cary, NC).
Histopathology of Breast Cancer Study Tissues
After staining but before assessment of ER or PR status, the tissue specimens were evaluated to ensure that they contained an adequate amount of invasive breast carcinoma tissue to obtain a reliable ER or PR status and acceptable morphology. Tissues not meeting these criteria were excluded from the study. For the ER and PR reproducibility studies, all 20 specimens in each study were acceptable and were included. Of the 250 specimens in the ER method comparison study, 11 were excluded due to inadequate invasive breast carcinoma tissue, and one due to retraction artifacts. Of the 300 specimens in the PR method comparison study, 8 were excluded due to inadequate invasive breast carcinoma tissue, and 3 due to unacceptable artifacts.
Overall Staining Quality
Invasive tumor cells across all tissues in these studies showed a spectrum of staining characteristics that ranged from an absence of ER/PR staining (IHC negative) to nuclear staining in a few cells (IHC positive if nuclear staining was present in >1% of invasive tumor cells) to diffuse strong nuclear reactivity across the tumor, which was comparable between the Dako Omnis and the Autostainer Link 48 staining platforms. Normal breast elements, when present in the section with invasive tumor, showed variable nuclear staining in virtually all cases regardless of the staining platform.
The specimens included in the studies were stratified to ensure that they represented the range of ER or PR expression encountered in clinical settings. The proportion of specimens in each study in various expression categories is shown in Table 2. The reproducibility studies included approximately 20% to 25% of specimens with invasive carcinoma around the 1% cutoff, and 11.8% (ER) and 14.2% (PR) for the method comparison studies.
Representative examples of the range of nuclear staining in invasive breast carcinoma tumor cells using the anti-ERα clone EP1 and anti-PR clone PgR 1294 antibodies on the Dako Omnis platform are shown in Figures 1 and 2. Occasional, scattered weak nuclear, and/or cytoplasmic staining of non-neoplastic cells including stromal cells, lymphocytes, and other cells was observed, and was similar between the 2 staining platforms (data not shown).
Across the 4 studies approximately 1% of the slides stained with the negative control reagents showed weak nuclear staining of either single or a very few invasive tumor cells but all were judged as acceptable. No slides stained with the negative control antibodies had any cytoplasmic staining of invasive tumor cells or cytoplasmic staining of nontumor cells (data not shown).
Results From the ER studies
In the method comparison study, sections from 250 tissue blocks were stained at 2 centers (IEO and URMC; 125 specimens each) with the anti-ERα clone EP1 antibody on the 2 staining platforms (Dako Omnis or Autostainer Link 48), and the percent agreement in ER status (positive or negative) was compared. The reproducibility study looked at interrun, interlaboratory, and interobserver reproducibility on sections from 20 tissue blocks stained with the anti-ERα clone EP1 antibody on the Dako Omnis platform in 5 runs at 3 centers (UCL, EIO, and URMC). The results from these studies are summarized in Table 3. High agreement rates were obtained for all study endpoints.
ER Staining Quality With Dako Omnis
The ER staining observed with the Dako Omnis platform showed crisp nuclear expression that varied in terms of the proportion of positive tumor cells and the intensity of signal across the different cases in this study (Fig. 1). Cytoplasmic staining in tumor cells was seen only in cases where there was diffuse strong nuclear staining present in the breast carcinoma.
For each ER-stained invasive carcinoma specimen included in the method comparison study, the pathologists recorded the percentage of positive cells and the staining intensity (Figure, Supplemental Digital Content 1, http://links.lww.com/AIMM/A101), as well as staining of DCIS components, if present on the section (Figure, Supplemental Digital Content 2, http://links.lww.com/AIMM/A102). The results were highly comparable between the 2 staining platforms in terms of the proportion of positive tumor cells, with a slight tendency for a higher staining intensity for cases run on the Dako Omnis platform compared with the Autostainer Link 48 platform. The pattern of DCIS staining was similar between the 2 staining platforms.
Results From the PR Studies
In the method comparison study, sections from 300 tissue blocks were stained at 3 centers (IEO, UCL, and URMC; 100 specimens each) with anti-PR clone PgR 636 antibody on the Autostainer Link 48 platform and anti-PR clone PgR 1294 antibody on the Dako Omnis platform and the percent agreement in PR status (positive or negative) was compared. The reproducibility study looked at interrun, interlaboratory, and interobserver reproducibility on sections from 20 tissue blocks stained with the anti-PR clone PgR 1294 antibody on the Dako Omnis platform in 5 runs at 3 centers (UCL, EIO, and URMC). The results from these studies are summarized in Table 4. High agreement rates were obtained for all study endpoints.
PR Staining Quality With Dako Omnis
The PR staining observed with the Dako Omnis platform also showed crisp nuclear expression that varied in terms of the proportion of positive tumor cells and the intensity of signal across the different cases in this study (Fig. 2). Cytoplasmic staining in tumor cells tended to be seen only in cases where there was diffuse strong nuclear staining present in the breast carcinoma.
For each PR-stained invasive carcinoma specimen included in the method comparison study, the pathologists recorded the percentage of positive cells and the staining intensity (Figure, Supplemental Digital Content 3, http://links.lww.com/AIMM/A103), as well as staining of DCIS components, if present on the section (Figure, Supplemental Digital Content 4, http://links.lww.com/AIMM/A104). The results were highly comparable between the 2 staining platforms in terms of the proportion of positive tumor cells, with a slight tendency for a higher staining intensity for cases run on the Autostainer Link 48 platform compared to the Dako Omnis platform. The pattern of DCIS staining was similar between the 2 staining platforms.
Accurate, reliable, and reproducible evaluation of the ER status in breast cancer is critically important to help ensure appropriate treatment planning.13 IHC is the main method currently used for this evaluation, and all laboratories that perform IHC assays for ER and PR should closely follow quality control and assurance measures as outlined in published guidelines.5,6,14 Breast cancer treatment is predicated on the ability of IHC to provide an accurate assessment of the expression of these biomarkers in formalin-fixed, paraffin-embedded breast cancer tumor tissue.5 Ideally, the assessment and interpretation of ER/PR staining would be standardized, to help ensure that results are reproducible between different observers and different laboratories regardless of reagents or staining platform used. However, a number of studies have documented significant discordant results as well as interlaboratory variability in ER assay results that have been attributed to a variety of causes, including sensitivity and specificity of antibody reagents,15–18 insufficient antigen retrieval,19–22 and differing threshold and interpretation criterion.20,23 These studies highlight the importance of rigorous and thorough evaluation of all aspects of testing, including technical validation of the performance of any new IHC staining platforms or new IHC reagents for ER and PR analysis before their clinical implementation.
In the present study we investigated new ER and PR assays and a novel automated staining platform in a large cohort of primary breast cancer cases obtained from at least 15 different hospitals or clinical sites, which would likely be representative of breast cancer cases seen in the general population across a number of different institutions and clinical sites. Our results show excellent concordance between the Dako Omnis staining platform compared with the existing marketed staining platform (Autostainer Link 48). There was excellent interrun, interobserver, and interlaboratory reproducibility for both ER (clone EP1) and PR (clone PgR 1294) antibodies employed on the Dako Omnis staining platform across the 3 different study laboratories. The staining results showed high specificity with crisp, distinct nuclear reactivity for both ER and PR antibodies both in tumor cells and normal luminal cells with very low background.
Although anti-ER clone EP1 and anti-PR clone PgR 1294 for Dako Omnis were not directly compared in the present studies to the Dako ER/PR pharmDx kit, indirect inference can be made based on the reported high degree of concordance (∼95%) between clone EP1 and the ER component of the pharmDx kit (a mixture of 2 mouse monoclonal antibodies) using the Allred scoring system to determine the cutoff for positivity.11 For detection of PR, the present study used clone PgR 1294 on the Dako Omnis, which is the same antibody used as the PR component of the pharmDx kit, but compared it with clone PgR 636 in order to consistently use the ASCO/CAP recommended cut-off of ≥1% positive tumor cells for positive assessment. Our results are consistent with those of Gill et al,24 who showed 96% agreement between anti-PR clone PgR 1294 and clone PgR 636.
For this study cohort, the proportions of specimens with invasive carcinoma around the 1% cutoff (derived from the actual percent-positive-cells scoring from the study data) was around 20% to 25% for the reproducibility studies with a mean of 11.8% (ER) and 14.2% (PR) for the method comparison studies, suggesting that this is a challenging and robust data set with which to make comparison between different staining platforms and antibody reagents. As has been previously described, the overall concordance would be expected to be higher in unequivocally positive or negative cases,25 whereas tumors in the low/borderline steroid hormone receptor group would be more challenging to reach a high degree of concordance.21 In a recent report among 1700 consecutive cases of invasive ductal carcinoma from a single institution, only 32 (1.9%) of cases fell into the ER positive 1% to 10% category,26 suggesting that the number of borderline cases in the current study is overrepresented compared with the general breast cancer population. In the cohort of breast cancer cases presented here, a high degree of concordance was demonstrated between anti-ER clone EP1 and anti-PR clone PgR 1294 used on Dako Omnis and the existing anti-ER and anti-PR Dako products for Autostainer Link 48. In addition, excellent reproducibility of the new anti-ER and anti-PR reagents was observed on the Dako Omnis across multiple runs, testing laboratories, and evaluating pathologists (observers).
The ability to detect a range of levels of expression of ER and PR, particular for cases with low levels of expression, is important and desirable for any biomarker assay for hormone receptors and may have prognostic significance. In a retrospective study of 1424 consecutive patients with HER2-negative breast cancer and low endocrine receptor expression, patients with ER/PR of 1% to 10% had a slight but not statistically significant, better prognosis than the ER/PR <1% group.27 These authors concluded that further studies are needed to identify the appropriate clinical approach in this subset of patients with low ER/PgR expression (ER/PgR 1% to 10%), HER2-negative early breast cancer. Thus, the accurate assessment of ER/PR, particularly for cases with low levels of expression, remains important for both treatment planning and furthering our understanding of the biology of breast tumors with different levels of ER/PR expression.26
The data presented here suggests that immunohistochemical analysis of ER and PR expression levels using monoclonal antibody clones EP1 and PgR 1294 on the Dako Omnis automated staining platform is reliable and reproducible across staining runs, testing laboratories, and evaluating pathologists.
The authors thank Francesca Ciocca, Marta Lisi, and Chiara Zanetti for their assistance in conducting the studies; Anette Fält for planning and coordination; Karen Jakobsen and Linda B. Jensen for discussions and input to the study designs; and Mary C. Jackson for editorial assistance and preparation of the manuscript.
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