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Originally published as JCO Early Release 10.1200/JCO.2009.23.3064 on May 26 2009 © 2009 American Society of Clinical Oncology.
Reply to V. Arena et alDepartment of Pathology, University Medical Center Hamburg-Eppendorf, University of Hamburg, Hamburg, Germany
Health Quality Research, Altarum Institute, Ann Arbor, MI
Edinburgh Cancer Research Centre, Western General Hospital, Edinburgh, United Kingdom
Division of Hematology and Oncology, Department of Medicine, Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA
Department of Pathology and Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA We are grateful for the interest Arena et al1 show in the issues related to human epidermal growth factor receptor 2 (HER2) testing as discussed in our article. We think the points they raise related to what quality requirements are appropriate and what costs in molecular diagnostics are important. We respond specifically to the points raised in their correspondence as follows: Petersen et al2 systematically address the technical issues related to fluorescen in situ hybridization (FISH) and illustrate the challenges that arise with variation of steps in the procedure. In contrast to Petersen et al, none of us would consider hybridization in as few as 50% to 75% of specimen nuclei to be acceptable hybridization levels. We routinely expect hybridization in nearly all nuclei (> 95%) before considering the sample as technically adequate for evaluation. Nevertheless, none of the technical issues identified by Petersen et al detract from the important advantages of FISH compared with immunohistochemistry (IHC). Both FISH and IHC have technical challenges that need to be addressed for successful performance of these assay methods. However, the technical issues related to FISH do not provide either a false-positive or a false-negative result, provided that tumor cell nuclei and not benign reactive cell nuclei present in the specimen have been scored. The number of gene copies per nucleus can be directly identified and counted by FISH. As mentioned in our article,3 an unsuccessful FISH assay yields a nonresult, not a false result. As briefly reviewed in our article and reported elsewhere,4–17 IHC is associated with a variable number of both false-positive and false-negative immunostaining results depending on how the specimen was fixed or processed for IHC. It is not clear to which "diagnostic pitfalls" Arena et al1 refer. The Dowsett et al18 study is a slide exchange study of only 20 different specimens that were deliberately selected to include a relatively high proportion of equivocal cases scored at five different laboratories. None of the diagnostic criteria that were used to assess HER2 status by FISH in "equivocal cases" (presumably FISH ratios between 1.80 and 2.20) were described. Therefore, we assume that the criteria for evaluation of equivocal cases were not standardized and would be expected to lead to differences of opinion about the HER2 status in different laboratories. The concordance papers that we cited were either studies of 100 or more unselected breast cancer specimens analyzed in individual laboratories or were studies of proficiency testing involving more than 100 different laboratories where the number of "equivocal cases" (cases near the FISH cutoff ratio of 2.0) would be expected to be relatively small (approximately 1% to 2%). Enrichment of an analyzed cohort for cases near the FISH ratio of 2.0 without a clear-cut definition of the criteria to be used for assessment of HER2 status among these specimens is expected to lead to discrepancies as observed in the Dowsett et al paper. If "only the combination of FISH evaluation with immunoistochemistry can reduce the possibility of diagnostic pitfalls"1 means that "it is important to note that by using both immunohistochemistry and FISH, as recommended in the HER2-testing algorithm, the chances of misdiagnosis are reduced,"18 then we disagree. As we summarize in our perspectives article,3 the use of IHC as a primary screen of all breast cancers with IHC 2+ breast cancers retested by FISH leads to a variable number of errors. Approximately 10% of all HER2-amplified breast cancers are either IHC 0 or IHC 1+, and women with these breast cancers respond significantly to HER2-targeted therapies with the same hazard ratio as women with IHC 3+/FISH-amplified breast cancers.19 Likewise, breast cancers that are HER2 IHC 3+ in a primary IHC screen, actually lack HER2 gene amplification in 5% to 22% of these cases and women with these breast cancers do not show a differential response to HER2-targeted therapy compared with patients with IHC 0/FISH-negative breast cancer.20 If Arena et al1 are advocating primary FISH testing and primary IHC testing of all breast cancers, we have no objection. However, our data indicate that IHC results contribute little useful addition information beyond the HER2 status determined by FISH. Both FISH and IHC require experienced technicians to perform the assay method as well as experienced pathologists to interpret the assay results. Although some laboratories may still perform IHC as an overnight procedure, most laboratories perform IHC as a 1-day procedure. FISH is an overnight procedure. We do not believe the extra half-day required for FISH delays patient treatment. The higher demonstrated accuracy of FISH compared with IHC17 is, in our opinion, well worth the additional time. What do Arenas et al1 mean by the suggestion that performing primary FISH testing for HER2 is not "really sustainable" in the "pathology laboratory economy"? They state that "using FISH as the primary test for HER-2 status determination, there would be an increment of the technical and medical daily workload and increased pathology lab-costs that could not be borne by all the peripheral laboratories."1 Our view is that if the FISH assay is not sufficiently remunerative in a particular laboratory setting to offset the greater expense of this assay method, or if the technical services of an experienced FISH laboratory are not available in a local hospital site, the patient's paraffin block could be shipped by overnight express mail to a reference laboratory that performs this assay and participates in a recognized quality assurance program. The slightly increased cost of FISH then becomes the problem of a reference laboratory, and the peripheral laboratory is relieved of this expense. In 2006, the United States alone had more than 139 laboratories participating in the HER2 FISH proficiency testing program of the College of American Pathologists.21 This is expected to be more than sufficient capacity to accommodate the needs for HER2 FISH testing. To our knowledge, no reference laboratory has been overwhelmed by the large volume of requests for this assay from referring laboratories. Nevertheless, our point remains: in the overall economy of the healthcare system, with the cost of the drugs and treatment of adverse effects in the equation, FISH remains more cost effective. Arenas et al1 express the concern that "finally, we believe that both the oncologists and pathologists, should not loose trust in immunohistochemistry at least as a primary assessment method of HER2 expression." We disagree. This is not about trust. This is about results. The data demonstrate that FISH is the more accurate and reproducible assay method. Primary screening of HER2 status by IHC excludes some women whose tumors are IHC 0/1+ but do have HER2 gene amplification from receiving HER2-targeted treatments, while it includes other women who have IHC 3+ breast cancers but lack HER2 gene amplification. In our opinion, our patients and their oncologists deserve no less than the best available HER2 testing method from their pathology laboratories and pathologists. It is worth the extra time, effort and expense. 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: None Consultant or Advisory Role: James Lee, Abbott Laboratories (C); Michael F. Press, Genentech, Inc (C), GlaxoSmithKline (C) Stock Ownership: None Honoraria: Guido Sauter, Abbott Laboratories; John M.S. Bartlett, Abbott Laboratories; Dennis J. Slamon, Aventis Pharmaceuticals, Genentech BioOncology, Roche Pharmaceuticals; Michael F. Press, Genentech Inc, GlaxoSmithKline Research Funding: Michael F. Press, Genentech Inc, GlaxoSmithKline Expert Testimony: None Other Remuneration: None REFERENCES
1. Arena V, Pennacchia I, Carbone A, et al: Fluorescent in situ hybridization for human epidermal growth factor receptor 2 assessment in breast cancer: Is it applicable as a primary test? J Clin Oncol 27:e8; 2009. 2. Petersen B, Sørensen M, Pedersen S, et al: Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue: Optimizing the method. Appl Immunohistochem. Mol Morphol 12:259–265, 2004. 3. Sauter G, Lee J, Bartlett J, et al: Guidelines for HER-2 testing: Biologic and methodologic considerations. J Clin Oncol 27:1323–1333, 2009. 4. Ciampa A, Xu B, Ayata G, et al: HER-2 status in breast cancer: Correlation of gene amplification by FISH with immunohistochemistry expression using advanced cellular imaging system. Appl Immunohistochem Mol Morphol 14:132–137, 2006.[CrossRef][Medline] 5. Dybdal N, Leiberman G, Anderson S, et al: Determination of HER2 gene amplification by fluorescence in situ hybridization and concordance with the clinical trials immunohistochemical assay in women with metastatic breast cancer evaluated for treatment with trastuzumab. Breast Cancer Res Treat 93:3–11, 2005.[Medline] 6. Ellis CM, Dyson MJ, Stephenson TJ, et al: HER2 amplification status in breast cancer: A comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques. J Clin Pathol 58:710–714, 2005. 7. Hammock L, Lewis M, Phillips C, et al: Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization. Hum Pathol 34:1043–1047, 2003.[CrossRef][Medline] 8. Hoang MP, Sahin AA, Ordonez NG, et al: HER-2/neu gene amplification compared with HER-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma. Am J Clin Pathol 113:852–859, 2000. 9. Hofmann M, Stoss O, Gaiser T, et al: Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin) phase II monotherapy study: Assessment of test sensitivity and impact of chromosome 17 polysomy. J Clin Pathol 61:89–94, 2008. 10. Lal P, Salazar PA, Hudis CA, et al: HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: A single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring. Am J Clin Pathol 121:631–636, 2004. 11. McCormick SR, Lillemoe TJ, Beneke J, et al: HER2 assessment by immunohistochemical analysis and fluorescence in situ hybridization: Comparison of HercepTest and PathVysion commercial assays. Am J Clin Pathol 117:935–943, 2002. 12. Perez EA, Roche PC, Jenkins RB, et al: HER2 testing in patients with breast cancer: Poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization. Mayo Clin Proc 77:148–154, 2002. 13. Press MF, Sauter G, Bernstein L, et al: Diagnostic evaluation of HER-2 as a molecular target: An assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials. Clin Cancer Res 11:6598–6607, 2005. 14. Roche PC, Suman VJ, Jenkins RB, et al: Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 94:855–857, 2002. 15. Simon R, Nocito A, Hubscher T, et al: Patterns of her-2/neu amplification and overexpression in primary and metastatic breast cancer. J Natl Cancer Inst 93:1141–1146, 2001. 16. Yaziji H, Goldstein LC, Barry TS, et al: HER-2 testing in breast cancer using parallel tissue-based methods. JAMA 291:1972–1977, 2004. 17. Press M, Slamon D, Flom K, et al: Evaluation of HER-2/neu gene amplification and overexpression: Comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 20:3095–3105, 2002. 18. Dowsett M, Hanna W, Kockx M, et al: Standardization of HER2 testing: Results of an international proficiency-testing ring study. Mod Pathol 20:584–591, 2007.[CrossRef][Medline] 19. Press M, Finn R, Cameron D, et al: HER2 gene amplification, HER2 and EGFR messenger RNA and protein expression and lapatinib efficacy in women with metastatic breast cancer. Clinical Cancer Res 14:7861–7870, 2008. 20. Mass R, Press M, Anderson S, et al: Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clinical Breast Cancer 6:240–246, 2005.[Medline] 21. Persons D, Tubbs R, Cooley L, et al: HER-2 fluorescence in situ hybridization: Results from the survey program of the College of American Pathologists. Arch Pathol Lab Med 130:325–331, 2006.[Medline]
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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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