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Journal of Clinical Oncology, Vol 26, No 15 (May 20), 2008: pp. 2433-2435 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.15.7800
Problems and Solutions in the Evaluation of Hormone Receptors in Breast CancerDepartment of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri An article by Badve et al1 in this issue of the Journal of Clinical Oncology describes an interesting study comparing two methods of assessing estrogen-receptor (ER) and progesterone-receptor (PR) expression in invasive breast cancer (IBC). One method measured protein by immunohistochemistry (IHC) and the other measured RNA using quantitative reverse-transcription polymerase chain reaction (RT-PCR). All assays utilized formalin-fixed, paraffin-embedded tissue samples from a case-control study cohort derived from the Eastern Cooperative Oncology Group clinical trial E2197, which randomly assigned patients with high-risk IBC to adjuvant doxorubicin/cyclophosphamide versus doxorubicin/docetaxel, with the addition of tamoxifen in receptor-positive cases.2 The cases (n = 179) represented all patients who relapsed (5 years follow-up) matched to an excess of controls (n = 597) who did not relapse, with available paraffin blocks. The IHC assays were performed twice in different laboratories. One was in the laboratories of institutions using local (and almost certainly diverse) staining protocols and definitions of positivity, most likely evaluating single 3- to 5-µm histologic sections from intact paraffin blocks of tumor. The other was in an expert central laboratory using a standardized staining protocol on single 3- to 5-µm sections from tissue microarrays (TMAs) containing two 1.0-mm cores from each tumor, with positive defined as an Allred Score of more than 2 (corresponding to 1% to 10% weakly positive cells).3,4 The RT-PCR assays were conducted with RNA extracted from multiple (three to six) 10-µm sections of intact paraffin blocks at Genomic Health Inc (GHI; Redwood City, CA) using the Oncotype DX assay,5 which evaluates 21 genes to determine a Recurrence Score (RS). The genes include ER and PR, with definitions of positive (6.5 and 5.5 units, respectively) determined in previous studies.5 Overall, there was a fairly high concordance of positive and negative results across all assays, ranging from 90% to 93% for ER, and 84% to 88% for PR. Considering a positive result by any assay to be truly positive, the discordant cases favored RT-PCR over central IHC for assessing ER (7.2% of all cases; 91% of RT-PCR positive), central IHC over RT-PCR for PR (10% of all cases; 81% of IHC positive), and discordances were evenly balanced comparing both receptors combined (ie, ER and/or PR positive = receptor positive; 6.6% of all cases). All ER and PR assays were strongly associated (P < .0001) with relapse in all patients combined, but only marginally in the subset of patients with ER-positive tumors (P = .0144 by RT-PCR and P = .0913 by IHC). However, the RS was a highly significant (P < .0001) predictor of relapse in tamoxifen-treated ER-positive patients, consistent with previous studies.5 In current medical practice, ER and PR status are critical in determining the use and estimating the benefit of adjuvant hormonal therapy, and there is a widely (and justifiably) held perception that IHC, the present standard methodology for measuring ER and PR status, is unreliable and inaccurate in a substantial proportion of cases.6,7 The importance of the Badve et al1 study stems from its investigation of alternative methods for evaluating receptors that may be more reliable and accurate. In the limited scope of this study, it was successful by demonstrating that RT-PCR is at least equivalent to IHC in its ability to identify receptor-positive cases (considering ER and PR combined), marginally superior in predicting outcome in ER-positive patients, and superior in technical precision, which are all encouraging results. There are many causes for the problems associated with measuring ER and PR by IHC.7 Some involve preanalytic issues unrelated to IHC itself, such as delayed fixation of tissue, which allows proteins to degrade. Others are analytic in nature, such as the use of diverse reagents with unequal sensitivities,4,8-10 or procedures which inadequately expose proteins masked during tissue fixation.7 Even postanalytic events may contribute, in the sense that tumors with low levels of receptors (eg, 1% to 10% positive cells) may respond to hormonal therapy,4,9,11 and some laboratories use arbitrary definitions of positive that are too high (eg, > 10% positive cells). With newer reagents and protocols, IHC assays for hormone receptors can even be too sensitive, resulting in bimodal results (negative or very high) that are inaccurate because breast cancers show a broad range of receptor expression.12-15 RT-PCR does not suffer this shortcoming and is able to more accurately convey this continuum. The central ER IHC results in the Badve et al1 study were essentially bimodal, suggesting that the assay was saturated, but also that insufficient sensitivity of the assay per se was not the reason that most of the discordant results were positive by RT-PCR but negative by IHC, although some of preanalytic issues alluded to could have resulted in false-negative IHC results. For example, perhaps some of the cases were poorly fixed and RT-PCR is better able to accommodate degraded RNA than IHC to accommodate degraded protein, although this has not been proven. Some of the discordances may have resulted from sampling error caused by the much smaller amount of tissue analyzed by IHC compared with RT-PCR in this study, which may have been inadequate to fully account for the heterogeneity of receptor expression in the tumors. Although some studies suggest that IHC performed on TMAs can closely approximate the results obtained by evaluating much larger tissue sections,16 they are still unlikely to account for all the heterogeneity, and TMAs are not used in routine clinical practice. Many of the problems with IHC mentioned above have solutions, but some do not. For example, most IHC assays rely on enzymatic detection systems with rapid kinetics that are very difficult to control, which is one of the main reasons they are so easy to saturate. In general, it is almost impossible for enzymatic IHC to generate highly reproducible quantitative results, although even this may be surmountable with newer immunofluorescence techniques.17 Some apparent problems, such as discordant results between IHC and RT-PCR, may not be problems at all in the sense that protein expression and RNA expression are not the same thing, and there is no a priori reason to expect similar results or performance in every setting. Nonetheless, assessing ER by RT-PCR in the Badve et al1 study was statistically superior to IHC in predicting relapse in tamoxifen-treated, ER-positive patients, which is a potentially significant achievement. The superiority of RT-PCR over IHC for measuring ER may indeed be generally true, but additional validation studies should be required before changing practice standards because the results were so close, the study design was imperfect to address this issue, and some IHC assays may perform better. Furthermore, receptor status in clinical practice today is not determined by ER alone, and a detailed comparison of the predictive abilities of PR alone, or ER/PR combined, was not provided in this study. On the basis of the concordance results, IHC performed better in assessing PR than ER, and may have fared better as a predictor of clinical outcome. Similar to IHC, RT-PCR is also difficult and fraught with potential problems, as Badve et al acknowledged. Although the assay is undoubtedly reliable and accurate in their hands, this may not be true in the hands of all of the thousands of laboratories around the world testing for ER and PR. Given the current need for accurate ER and PR results in all patients with breast cancer, an obvious short-term priority should be to improve the quality of widespread IHC testing, in addition to encouraging new studies intended to develop better tests, such as the laudable effort by Badve. Although enzymatic IHC is not well suited for analyses requiring a high degree of analytic precision or quantification, it is still very useful in many settings. In the setting of hormone receptors in breast cancer, a number of studies have demonstrated that the IHC procedures used in some laboratories are capable of providing results that are as reliable and useful as the previous gold standard ligand binding assays (LBAs), which it replaced, and LBAs are precise and highly quantitative4,8,9,11,18-20 (RT-PCR may be better than either, and only time will tell). Improving the quality of IHC assays on a global scale will require that all laboratories implement such rigorously standardized and validated assays, which will be difficult. The College of American Pathologists and the American Society of Clinical Oncology are aware of this need, and are initiating an effort to develop and publish enforceable guidelines for ER and PR testing, similar to recent College of American Pathologists/American Society of Clinical Oncology guidelines for human epidermal growth factor receptor 2 testing.21 Ultimately, however, it is unrealistic to expect that even perfect tests for ER and PR alone, regardless of the technology, will be sufficiently powerful to accurately predict the response of all patients because the biology is so complex. New, more powerful predictors are needed, most likely based on multiple biomarkers. In this regard, the Badve et al1 study provided further validation that the Oncotype DX RS is powerful in further stratifying response to hormonal therapy in receptor-positive patients, although including individual ER and PR results would be a welcome addition to the report. Other strategies under investigation also look promising, including the HOXB13/IL17BR gene ratio determined by RT-PCR,22-24 and estrogen-regulated gene signatures determined by microarrays.25 Hopefully, these and other approaches will lead to significant improvements in predicting response to hormonal therapies in the near future. AUTHOR'S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: D. Craig Allred, Genomic Health Inc (C) Stock Ownership: None Honoraria: None Research Funding: None Expert Testimony: None Other Remuneration: None REFERENCES
1. Badve S, Baehner FL, Gray R, et al: Estrogen- and progesterone-receptor status in ECOG 2197: Comparison of immunohistochemistry by local and central laboratories and quantitative reverse transcription polymerase chain reaction by central laboratory. J Clin Oncol 26:2473-2481, 2008 2. Goldstein L, O’Neill A, Sparano JA, et al: E2197:Phase III (AT (doxorubicin/docetaxel) vs AC (doxorubicin/cyclophosphamide) in the adjuvanat treatment of node positive and high risk node negative breast cancer. J Clin Oncol 23:512, 2005 (suppl; abstr XXXX) 3. Allred DC, Harvey JM, Berardo MD, et al: Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol 11:155-168, 1998[Medline] 4. Harvey JM, Clark GM, Osborne CK, et al: Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol 17:1474-1481, 1999 5. Paik S, Shak S, Tang G, et al: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. N Engl J Med 351:2817-2826, 2004 6. Mathews AW: Bad cancer tests drawing scrutiny. Wall Street Journal, 2008, pp B1-B2 7. Rhodes A, Jasani B, Balaton AJ, et al: Immunohistochemical demonstration of oestrogen and progesterone receptors: Correlation of standards achieved on in house tumours with that achieved on external quality assessment material in over 150 laboratories from 26 countries. J Clin Pathol 53:292-301, 2000 8. Cheang MC, Treaba DO, Speers CH, et al: Immunohistochemical detection using the new rabbit monoclonal antibody SP1 of estrogen receptor in breast cancer is superior to mouse monoclonal antibody 1D5 in predicting survival. J Clin Oncol 24:5637-5644, 2006 9. Mohsin SK, Weiss H, Havighurst T, et al: Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: A validation study. Mod Pathol 17:1545-1554, 2004[CrossRef][Medline] 10. Press M, Spaulding B, Groshen S, et al: Comparison of different antibodies for detection of progesterone receptor in breast cancer. Steroids 67:799-813, 2002[CrossRef][Medline] 11. Love RR, Duc NB, Allred DC, et al: Oophorectomy and tamoxifen adjuvant therapy in premenopausal Vietnamese and Chinese women with operable breast cancer. J Clin Oncol 20:2559-2566, 2002 12. Allred DC, Brown P, Medina D: The origins of estrogen receptor alpha-positive and estrogen receptor alpha-negative human breast cancer. Breast Cancer Res 6:240-245, 2004[CrossRef][Medline] 13. Allred DC, Mohsin SK: ER expression is not bimodal in breast cancer. Am J Clin Pathol 124:474-476, 2005[Medline] 14. Collins LC, Botero ML, Schnitt SJ: Bimodal frequency distribution of estrogen receptor immunohistochemical staining results in breast cancer: An analysis of 825 cases. Am J Clin Pathol 123:16-20, 2005[CrossRef][Medline] 15. Swanson PE, Schmidt RA: Beneath the surface of the mud, part II: The dichotomization of continuous biologic variables by maximizing immunohistochemical method sensitivity. Am J Clin Pathol 123:9-12, 2005[CrossRef][Medline] 16. Zhang D, Salto-Tellez M, Putti TC, et al: Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer. Mod Pathol 16:79-84, 2003[CrossRef][Medline] 17. Chung GG, Zerkowski MP, Ghosh S, et al: Quantitative analysis of estrogen receptor heterogeneity in breast cancer. Lab Invest 87:662-669, 2007[CrossRef][Medline] 18. Barnes DM, Harris WH, Smith P, et al: Immunohistochemical determination of oestrogen receptor: Comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer 74:1445-1451, 1996[Medline] 19. Clahsen PC, van de Velde CJ, Duval C, et al: The utility of mitotic index, oestrogen receptor and Ki-67 measurements in the creation of novel prognostic indices for node-negative breast cancer. Eur J Surg Oncol 25:356-363, 1999[CrossRef][Medline] 20. Phillips T, Murray G, Wakamiya K, et al: Development of standard estrogen and progesterone receptor immunohistochemical assays for selection of patients for antihormonal therapy. Appl Immunohistochem Mol Morphol 15:325-331, 2007[CrossRef][Medline] 21. Wolff AC, Hammond ME, Schwartz JN, et al: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25:118-145, 2007 22. Goetz MP, Suman VJ, Ingle JN, et al: A two-gene expression ratio of homeobox 13 and interleukin-17B receptor for prediction of recurrence and survival in women receiving adjuvant tamoxifen. Clin Cancer Res 12:2080-2087, 2006 23. Ma XJ, Hilsenbeck SG, Wang W, et al: The HOXB13:IL17BR expression index is a prognostic factor in early-stage breast cancer. J Clin Oncol 24:4611-4619, 2006 24. Ma XJ, Wang Z, Ryan PD, et al: A two-gene expression ratio predicts clinical outcome in breast cancer patients treated with tamoxifen. Cancer Cell 5:607-616, 2004[CrossRef][Medline] 25. Oh DS, Troester MA, Usary J, et al: Estrogen-regulated genes predict survival in hormone receptor-positive breast cancers. J Clin Oncol 24:1656-1664, 2006
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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