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Originally published as JCO Early Release 10.1200/JCO.2009.21.8685 on April 20 2009 © 2009 American Society of Clinical Oncology.
Reply to J.M. Guinebretiere and L. Arnould et al
Indiana University, Bloomington, IN; Eastern Cooperative Oncology Group, Boston, MA
University of California, San Francisco, San Francisco; Genomic Health Inc, Redwood City, CA
Eastern Cooperative Oncology Group, Boston, MA
sanofi-aventis, Bridgewater, NJ
Genomic Health Inc, Redwood City, CA
Sanofi-aventis, Bridgewater, NJ
Genomic Health Inc, Redwood City, CA
North Central Cancer Treatment Group, Rochester, MN
Cancer and Acute Leukemia Group B, Chicago, IL
Southwest Oncology Group, Ann Arbor, MI
Eastern Cooperative Oncology Group, Boston, MA We thank Dr Guinebretiere1 and Arnould et al2 for their interest in our article regarding the assessment of estrogen receptor (ER) and progesterone receptor (PR) status in Eastern Cooperative Oncology Group (ECOG) 2197 using immunohistochemistry (IHC) by local and central laboratories and quantitative reverse transcription polymerase chain reaction (qRT-PCR) by a central laboratory.3 Collectively these letters raise several issues that are of significance and are briefly addressed here. We agree that technical choices are critical in performing IHC assays, and these concerns are why the American Society of Clinical Oncology/College of American Pathologists are developing guidelines for ER and PR testing, ones similar to those already published for human epidermal growth factor receptor 2 testing.4 To minimize confounding sources of analytic variability we used a central laboratory to rigorously perform IHC. ECOG's Pathology Core Laboratory performed the IHC. This laboratory is certified under Clinical Laboratory Improvement Amendments and performs all IHC assays for ECOG clinical trials using the assistance of automated instruments. The ECOG laboratory analytic process, including details about the primary antibodies, the commercially available Dako Envision+ kit (Dako, Carpinteria, CA)—a highly sensitive polymer amplification system—and the use of positive and negative controls, were detailed in the article.3 The expression of ER using 1D5 or 6F11 antibodies has been extensively studied, as have the correlates with the ligand-binding assay and with therapeutic benefit. This extensive clinical validation experience favored the used of 1D5 over the new ER clone, SP1. With regard to the methods for reading IHC, the interpretation of all cases was systematically performed by two experienced breast histopathologists, in tandem, and consensus was reached on all cases, using the semiquantitative Allred Score (assesses both intensity and percentage positive cells5). Characteristics of the discordant patient cases have been analyzed in greater detail. Twenty-five patient cases were ER negative by RT-PCR and were ER positive by IHC (either local or central or both local and central). One of the 25 patient cases was positive by both central and local IHC. Twenty-four of the 25 patient cases were positive by only local or only central, but not by both. Seventy-two cases were ER positive by RT-PCR and ER negative by IHC (either local or central or both local and central). Twenty-two of the 72 cases were negative by both central and local IHC. Fifty of the 72 cases were negative by only local or only central, but not by both. These results suggest that many of the discordances do not reflect real differences between RNA measurement and protein measurement but may be attributed to inter-laboratory variability in IHC assessment. The RT-PCR cutoff points for ER and PR were developed from three prior studies comparing RT-PCR with IHC.6–8 They were prespecified for this study and for the Habel et al9 study. The Habel et al study used the ER rabbit monoclonal antibody SP1, using tumor whole sections and optimized IHC assays that were performed in a high-volume commercial laboratory, and a high concordance between RT-PCR using Oncotype DX (Genomic Health, Redwood City, CA) and central IHC was again observed.10 The clinical significance of the prespecified ER and PR cutoff points used in these studies were furthermore examined by the National Surgical Adjuvant Breast and Bowel Project (NSABP) and Genomic Health in NSABP B-14 and were found to be of clinical significance with regards to tamoxifen benefit.11 One of the strengths of gene expression assessment using quantitative RT-PCR from fixed paraffin-embedded tissue is that, unlike previous biochemical methods of ER and PR using the ligand-binding assay, morphologic review of the hematoxylin and eosin–stained section is possible. In one third of all cases submitted for RT-PCR analysis, manual microdissection to enrich the invasive carcinoma is performed, using the hematoxylin and eosin slide as a guide.12 The requisite tumor required for this assay has been established by both technical feasibility studies13 and by rigorous clinical validation of the assay in more than 4,000 patients.9,14,15 The failure rate in the Genomic Health Clinical Laboratory, which has processed more than 85,000 samples from 40 countries, has been consistently less than 3%. Using IHC, the issue of biologic heterogeneity has been complicated by sources of preanalytical variability.16 The RT-PCR assay used in the study has been optimized for performance in fixed paraffin-embedded tissues of varying ages and is robust with regard to all sources of preanalytical variability. Additionally, the use of five reference genes allows for normalization, and the use of RNA standards to qualify reagents, machines, operators, and daily results addresses these sources of preanalytic variability by a method that is unique to RT-PCR.13 Thus, the observed degree of biologic variation using RT-PCR has been shown to be minimal.17 It should be noted that the manuscript documents near equivalency of the two analytic methods (IHC v RT-PCR) for determining ER and/or PR expression. Since all patients in the study population received chemotherapy (plus endocrine therapy if hormone receptor–positive determined locally), our study cannot address the question as to whether IHC or quantitative RT-PCR using Oncotype DX more accurately predicts benefit from endocrine therapy (which would have required a study population randomized to receive endocrine therapy or not). Although the Oncotype DX does result in added cost of diagnostic testing, both American Society of Clinical Oncology and National Comprehensive Cancer Network guidelines recommend that the assay be considered in appropriately selected patients. When a clinician chooses to order the Oncotype DX assay in addition to the Recurrence Score, quantitative single-gene results for ER, PR, and human epidermal growth factor receptor 2 are provided without additional charge. Pathologists may choose to use the reported quantitative single gene information for quality control or quality assurance purposes. AUTHORS' 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: Frederick L. Baehner, Genomic Health Inc (C); Barrett H. Childs, sanofi-aventis (C); Tara Maddala, Genomic Health Inc (C); Mei-Lan Liu, Genomic Health Inc (C); Steve C. Rowley, sanofi-aventis (C); Steven Shak, Genomic Health Inc (C) Consultant or Advisory Role: Joseph A. Sparano, Genomic Health Inc (C) Stock Ownership: Frederick L. Baehner, Genomic Health Inc; Steven Shak, Genomic Health Inc Honoraria: None Research Funding: None Expert Testimony: None Other Remuneration: None REFERENCES
1. Guinebretiere JM: Cancer is heterogeneous. J Clin Oncol 27:2732; 2009. 2. Arnould L, Coudert B, Fumoleau P: Is immunohistochemistry less sensitive than quantitative reverse transcription polymerase chain reaction for hormone receptors status determination in breast cancer? J Clin Oncol 27:2733; 2009. 3. Badve SS, Baehner FL, Gray RP, et al: Estrogen- and progesterone-receptor status in ECOG 2197: Comparison of immunohistochemistry by local and central laboratories and quantitative reverse-transcriptase polymerase chain reaction by central laboratory. J Clin Oncol 26:2473–2481, 2008. 4. 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 17159189. 5. 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 10334533. 6. Chang JC, Makris A, Gutierrez MC, et al: Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients. Breast Cancer Res Treat 108:233–240, 2008 17468949.[CrossRef][Medline] 7. Cobleigh MA, Tabesh B, Bitterman P, et al: Tumor gene expression and prognosis in breast cancer patients with 10 or more positive lymph nodes. Clin Cancer Res 11:8623–8631, 2005 16361546. 8. Mina L, Soule SE, Badve S, et al: Predicting response to primary chemotherapy: Gene expression profiling of paraffin-embedded core biopsy tissue. Breast Cancer Res Treat 103:197–208, 2007 17039265.[CrossRef][Medline] 9. Habel LA, Shak S, Jacobs MK, et al: A population-based study of tumor gene expression and risk of breast cancer death among lymph node-negative patients. Breast Cancer Res 8:R25; 2006.[CrossRef][Medline] 10. Baehner FL, Maddala T, Alexander C, et al: A Kaiser Permanente population-based study of ER and PR expression by standard method, immunohistochemistry (IHC), compared to a new method, quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Mod Pathol 21:20A; 2007 abstr 81. 11. Paik S, Shak S, Tang G, et al: Expression of the 21 genes in the Recurrence Score assay and tamoxifen clinical benefit in the NSABP study B-14 of node-negative, estrogen receptor–positive breast cancer. J Clin Oncol 23:16s; 2005. 12. Baehner FL, Hiller B, Kim C, et al: Use of macrodissection in multi-gene RNA analysis of fixed paraffin embedded tissue. Mod Pathol 17:22A; 2004 abstr 80.[CrossRef] 13. Cronin M, Pho M, Dutta D, et al: Measurement of gene expression in archival paraffin-embedded tissues: Development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay. Am J Pathol 164:35–42, 2004 14695316. 14. 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 15591335. 15. Paik S, Tang G, Shak S, et al: Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor–positive breast cancer. J Clin Oncol 24:3726–3734, 2006 16720680. 16. Goldstein NS, Ferkowicz M, Odish E, et al: Minimum formalin fixation time for consistent estrogen receptor immunohistochemical staining of invasive breast carcinoma. Am J Clin Pathol 120:86–92, 2003 12866377. 17. Baehner FL, Baker J, Salter J, et al: Heterogeneity of quantitative RT-PCR measurement of estrogen and progesterone receptor expression: Comparison of tissue microarray cores to whole sections of paraffin embedded breast cancer tissue. Mod Pathol 21:21A; 2008 abstr 83.
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Copyright © 2009 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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