Section 1 -

Hormone Receptor Analysis of Breast Cancer: Current Issues Nour Sneige University of Texas M.D. Anderson Cancer Center Houston, TX

Abstract: The estrogen receptor (ER) is a regulator of cellular growth, proliferation, and differentiation. In addition to having prognostic value, ER is the most important biologic marker of therapeutic response in breast cancer. Some level of measurable ER protein is expressed in 70-80% of human breast. Immunohistochemistry is the current method of choice for ER assessment and its predictive value has been shown to be superior to that of biochemically based assays. Although accurate ER protein assessment is critical for optimal treatment of patients with breast cancer, studies have demonstrated interlaboratory variability in ER detection. False-negative results for tumors with low ER protein levels have been a subject of recent concern. Lack of standardization for immunohistochemistry between laboratories is thought to be the major reason for testing errors although variability in scoring methods and reporting practices, which can affect results, also plays a role. In this presentation, I will discuss studies addressing interlaboratory variability and recommend optimal testing techniques and reporting procedures for ER testing, with the goal of increasing interlaboratory standardization for ER analysis by immunohistochemistry. Discussion: ER is a member of the family of nuclear receptors and functions as a transcriptional regulator. Currently, two different major ER proteins are known to exist, ERα and ERβ. Because ERβ does not currently have a role as a clinically useful breast tumor marker, it will not be covered in this presentation. Normal breast epithelium expresses ER to varying degrees. Benign ducts and lobules display patchy to diffuse ER expression and ductal epithelium tends to contain higher expression levels compared to lobular epithelium. ER expression levels in the normal breast are thought to remain relatively stable over time, gradually increase with age, and increased expression is associated with increased breast cancer risk. The follicular phase of the menstrual cycle has been associated with increased ER expression in the breast, although the results of a recent study did not confirm this finding. As many as 80% of invasive breast cancers express ER, and rates of expression increase with patient age and following menopause. Individual tumors typically express ER in a relatively homogenous fashion, although staining heterogeneity can be observed in a subset of cases. ER is a weak positive prognosticator for breast cancer whose effects on outcome can be observed for over a decade. More importantly, ER is a strong predictor of clinical response to hormonal therapies and the best response is seen in patients whose tumors express both ER and progesterone receptor. Tumors may also be solely ER-positive (relatively common) or progesterone receptor-positive (observed infrequently). Until the late 1990s, ER status was assessed in fresh (tumor) tissue using ligand binding assay. Recently, however, IHC has become the method of choice for determining ER status. Studies comparing IHC to the conventional biochemical assays have demonstrated that IHC is equivalent or superior to ligand binding assay for predicting response to hormonal therapy. Although IHC is a technique used by most pathology laboratories, concern exists over high rates of potentially false-negative results in ER testing. It is thought that false negative results are largely due to poor interlaboratory standardization for IHC and specifically to a lack of validated testing methods for ER specifically. Additional sources of variability include the choice of antibody, antigen retrieval techniques, use of controls, interpretation and scoring practices, and pre-analytical variables. The results of a recent laboratory survey of ER testing practices (Rhodes et al, and Layfield et al) highlighted these differences between individual laboratories for ER testing standards, and considerable variability for several of these parameters was documented. Rhodes et al, found only 37% of the participating laboratories were able to obtain a positive result for the presence of ER in tumors with low ER levels using the traditional 10% staining cutoff but 66% reported a positive result if a 1% cutoff was used. Their findings also strongly suggested that preanalytical variables (tissue handling, fixation and processing) do not greatly affect ER testing results using IHC. In a later report, the length of time for heat antigen retrieval was identified as the most important variable for improving ER testing standardization. Additional interlaboratory comparisons of ER testing performed in Austria and Sweden addressed staining technique and scoring reproducibility, respectively. Although variation was demonstrated in both of these studies, the authors concluded that improvements in testing could be made through automation and training. ER testing findings for intraductal carcinoma from NSABP Protocol B-24 have recently been presented by Allred et al. The predictive value of a positive ER status for response to tamoxifen therapy was demonstrated by these data. Additionally, it was observed that cases analyzed by participating institutions using non-standardized methods were more frequently ER negative compared to those tested by a centralized IHC laboratory (where a clinically validated and standardized testing method was utilized). Technical Considerations: When to Test All primary invasive breast carcinomas. Ductal carcinoma in- situ. Recurrent and/or metastatic breast cancer (when a change of ER status would affect treatment decisions) because of potential alterations of the ER status of tumors over time. Types of Specimens: Formalin-fixed, paraffin-embedded histologic tumor sections. Analysis of ER in smaller-sized, paraffin-embedded specimens (such as needle biopsies) and air-dried or alcohol fixed direct smears can also be performed. Tissue Handling, Fixation and Processing: Breast tumors should be placed in fixative within one hour from the time of removal from the patient. The ideal tumor thickness in tissue cassettes is 2-4 mm. The standard fixative is traditionally 10% neutral buffered formalin. Alcohol-based fixatives (eg alcoholic formalin) may be utilized. For testing of metastatic disease of bone, our experience indicates that decalcification solutions and mercury containing fixatives (eg B5) can affect ER antigenicity and produce false negative results. The minimum fixation time needed to obtain optimal and consistent ER staining results is 6-8 hours for all paraffin-embedded specimens. Overfixation can markedly reduce ER positivity in some cases (Goldstein). Long-term storage of unstained paraffin-embedded sections can also alter ER immunoreactivity of breast tumors. For control tissues it is advisable to cut sections within 1 week of use for testing. Antibodies for ER: The followings are the most sensitive monoclonal antibodies (when used in combination with antigen-retrieval procedures) for ER detection. Antibody Source Manufacturer Dilution Ranges 1D5 Mouse Dako Cytomation 1:10 - 1:50 6F11 Mouse Novocastra Laboratories Ltd. 1:20 - 1:50 SP1 Rabbit Lab-Vision-NeoMarkers 1:30 - 1:100 Staining Methods: Most pathology laboratories performing IHC routinely use automated stainers. The use of automation does not totally standardize staining procedures, however, and factors that can contribute to variability of results still exist. Antigen Retrieval: Antigen retrieval is one of the most important factors for achieving accurate and consistent results for ER testing by IHC for both histologic and cytologic material. The recommended antigen retrieval time for ER-directed IHC ranges from 15 to 45 minutes. It is importantly that individual laboratories determine the optimal antigen retrieval time for ER testing. In the NEQAS-ICC study, extending the antigen retrieval times to 25 minutes provided the most consistent results between participating laboratories regardless of the buffer, buffer pH, or heating mechanism used. Quality Control: Controls are integral for laboratory-based testing and should be included in every immunohistochemical run for each marker tested antigen controls. Internal controls can be extremely useful for evaluating the quality of IHC. For ER testing in breast cancer cases, adjacent benign ducts and lobules make excellent internal comparators for the lesion in question. Quality Assurance: Establishment of rigorous quality assurance programs for ER testing and other IHC assays are currently under way in Europe and the United States. Quality assurance programs (such as United Kingdom's NEQAS-ICC or the College of American Pathologists proficiency programs for IHC) will play integral roles in the further development of formal quality assurance guidelines for ER testing. Scoring: Multiple scoring methods have been advocated for ER IHC and scoring cutoffs utilized for laboratories have been shown to range from 1% to 30%. No study, however, has convincingly demonstrated the clinical importance of measuring (or attempting to quantify) ER staining intensity or heterogeneity. The commonly applied 10% cutoff was establish by the report of Pertschuk et al. who demonstrated a response to treatment in patients with metastatic disease whose paraffin-embedded breast tumors were assayed with 1D5. Studies using 6F11 have demonstrated that an Allred score of 3 or more is sufficient to predict response. An Allred Score of 3 includes cases with 1/100th proportion of tumor cells expressing ER and moderate intensity (2+) nuclear staining. In our experience, these low level ER expressing tumors represent less than 5% of all breast cancer cases. Recent investigations have shown that manual scoring results were very similar to those obtained through image analysis and that the various analysis systems used were comparable. Reporting: The ER reporting system advocated by the International Breast Cancer Study Group is a three-tiered categorization of ER staining percentages that acknowledges the existence of both "positive" and "low positive" cases. Both the College of American Pathologists and the American Society of Clinical Oncology have issued consensus panel statements supporting the use of this approach, in which staining percentages as low as 1% are reported. Recommended categories for classification of ER staining results are shown below. % Positively Stained Tumor Nuclei Classification 0 Negative 1-9 Low Positive 10-100 Positive References: Diaz LK, Sneige N. Estrogen receptor analysis for breast cancer: Current issues and keys to increasing testing accuracy. Adv Anat Pathol 2005;12:10-19. Elledge RM, Green S, Pugh R et al. Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer: a Southwest Oncology Group Study. Int J Cancer. 2000;89:111-7. Fitzgibbons PL, Page DL, Weaver D, et al. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med. 2000;124:966-78. Goldhirsch A, Glick JH, Gelber RD, et al. Meeting highlights: International Consensus Panel on the Treatment of Primary Breast Cancer. 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