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Journal of Clinical Oncology, Vol 25, No 25 (September 1), 2007: pp. 4021-4023 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.11.4363
In ReplyThe Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD
Intermountain Healthcare, University of Utah School of Medicine, Salt Lake City, UT
Presbyterian Hospital, Charlotte, NC
University of Michigan Comprehensive Cancer Center, Ann Arbor, MI On behalf of the ASCO/CAP Panel on HER-2 Testing in Breast Cancer In his letter, Mr Raji describes some of the challenges faced when adopting new technologies in routine clinical practice. The American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) Panel on HER-2 [human epidermal growth factor receptor 2] Testing in Breast Cancer is well aware that there are significant preanalytic issues related to breast specimen processing that can affect the accuracy of HER-2 testing. All US Food and Drug Administration (FDA) –approved HER-2 testing kits (protein expression and gene amplification) describe only formalin fixation and large tissue samples were used to determine optimal fixation times. As the HER-2 testing guideline recommendations are an attempt to standardize handling conditions, any variance from these suggested times must be validated by laboratories using comparison with formalin-fixed samples fixed for between 6 and 48 hours. Although formalin penetrates tissues at a rate of 1 mm/h, the minimum fixation time for core biopsy specimens is not known, and the Panel did not find sufficient information about fixation durations that merited a specific recommendation. Laboratories and investigators evaluating the effects of longer and shorter fixation times and other methods (eg, the use of microwave fixation) are encouraged to submit their findings to the peer-review process and share them with the ASCO/CAP Panel. We hope that the publication of a guideline document on HER-2 testing will elicit more information about the effect of these variables on tissue HER-2 expression and that this will be made available to inform future revisions of the guideline document. The ASCO/CAP Panel on HER-2 Testing in Breast Cancer also wishes to thank Dr Nielsen and colleagues at Dako for reminding us of the May 2005 US FDA approval of the DakoCytomation HER2 FISH (fluorescence in situ hybridization) pharmDx Kit (Dako Denmark A/S, Glostrup, Denmark) for the assessment of patients being considered for trastuzumab therapy, and as an adjunct, to estimate prognosis in lymph node–positive breast cancer (www.fda.gov/cdrh/mda/docs/p040005.html). We also appreciate the opportunity to further expand on the guideline recommendation to refine the immunohistochemistry (IHC) criteria used to classify a tumor as IHC 3+ or HER-2 positive, which was based on expert panel consensus guided by a systematic review of the available evidence. The revised criterion for IHC 3+ using a validated IHC assay for HER-2 protein now recommends that more than 30% (instead of > 10%) of invasive breast tumor cells show uniform intense membrane staining.1,2 As detailed in Appendix G of the HER-2 testing guideline, "For IHC assays of HER2 protein expression, the original US FDA-approved interpretation guidelines provide insufficient specificity. Several experts, including those serving as central reviewers on clinical trials, have specified that a threshold of more than 30% of tumor (rather than the originally specified 10%) should show strong circumferential membrane staining for a positive result. This means that according to this guideline, strong circumferential staining of 30% or less of cells would be considered equivocal and be subjected to confirmatory FISH testing. A cutoff of more than 30% reflects the cumulative experience of panel members that usually a high percentage of the cells will be positive if it is a true IHC3+, published reports using cutoff values higher than 10%,3" and the goal of the panel to recommend reflex FISH testing to further characterize specimens that fall within these IHC boundaries. It is important to remember that the 10% criteria for HER-2 assessment in the Clinical Trials Assay in the initial metastatic trastuzumab studies was chosen for technical (not clinical) reasons as a way to deal with uncontrollable fixation variability that could lead to abrupt changes in HER-2 IHC pattern from one slide section to another.4 We stated on page 126 of the Journal that "Very rarely, in the experience of panel members, invasive tumors can show intense, complete membrane staining of 30% or fewer tumor cells. These are also considered to be equivocal in this guideline. Some but not all of these samples may have HER2 gene amplification and require additional testing to define the true HER2 status" (Fig 1 [shown] and Fig 2 of the Guidelines1).5,6
In summary, we believe that both data and expert experience strongly suggest that intra- and interobserver ability to reliably and reproducibly determine "IHC 3+" is very consistent above the 30% cutoff, but very inconsistent in the 10% to 30% range. Thus, increasing the cutoff will result in more uniform reading of the IHC 3+ category, and move a few patients into the IHC 2+ group for whom reflex FISH is appropriate. This change in the definition of IHC 3+ was driven by the Panel's recommendation that laboratories show 95% concordance with another validated IHC assay for both positive and negative results before adopting any assay as the primary or sole method of determining HER-2 status. To achieve that threshold, the Panel concluded that cases with diffuse complete membrane staining in 30% or less of invasive tumor cells are much less likely to have gene amplification and should now be described as IHC 2+. A HER-2 equivocal result will still trigger reflex testing using a validated in-situ hybridization assay, while allowing laboratories to achieve the desired internal levels of testing concordance. The Panel is aware that several of the definitive randomized adjuvant trastuzumab trials defined tumors as IHC 3+ (HER-2 positive) if more than 10% of invasive tumor cells showed uniform intense membrane staining using the Herceptest (Dako Denmark A/S, Glostrup, Denmark) assay done in a central or reference laboratory.7,8 It is unknown whether trastuzumab is or is not effective in patients who fall into this subset of being classified "IHC 2+" by virtue of having 10% to 30% cells positive and whose FISH tests are negative. Concern has been expressed that this fraction of patients, who were eligible for and participated in the randomized trials, would now be arbitrarily denied therapy with trastuzumab. The size of this fraction is unknown, so we queried investigators who led the North American Intergroup N9831 and those who led the National Surgical Adjuvant Breast and Bowel Project (NSABP) B31 trials. Limited data, thus far released only as part of Table 2 in the November 2006 revision of the trastuzumab package insert, show that 51 (3.5%) of 1,446 patients enrolled in Intergroup trial N9831 were ultimately found on central review to have IHC 3+/FISH-negative tumors9; however, data regarding the percentage of cells with diffuse complete membrane staining by IHC in these specimens are not yet available. A preliminary review of specimens from the NSABP B-31 trial suggests that the risk of not treating eligible patients would be small in laboratories performing high-quality HER-2 testing. Among approximately 50 patients who had tumors originally classified as IHC 3+ (HER-2 positive) who would now be reclassified as IHC 2+ (HER-2 equivocal) using the revised criterion, reflex FISH testing in about 43 patients would have shown gene amplification (personal communication, Soonmyung Paik, MD, March 25, 2007) and allowed them to still be considered candidates for therapy with trastuzumab. Thus, approximately seven of the more than 2,000 (0.35%) patients enrolled in trial B31 would have not been eligible using the revised IHC 3+ criteria.
The ASCO/CAP Panel has already addressed the issue of patients who by FISH, were found to have a HER2/CEP17 gene amplification ratio between 2.0 and 2.2. Even though FISH test results in this range are now considered HER-2–equivocal, Figure 2 of the guideline already reminds the reader that patients with a HER2/CEP17 ratio These issues highlight the existing uncertainty about the effects of anti-HER2 therapy in patients with discordant results (IHC-positive/FISH-negative, and vice versa) and in patients with tumors showing various degrees of HER2 expression and/or gene amplification. In view of the larger number of patients needed to populate these various HER-2 subsets and adequately power such retrospective analyses, it is critical that the leadership of all the adjuvant trastuzumab trials collaborate to attempt to answer these various questions, and the ASCO/CAP Panel has been in contact with the various groups to facilitate these efforts. In the meantime, available data suggest that high-quality HER-2 testing using a validated IHC or FISH assay is predictive of benefit from trastuzumab. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author(s) indicated no potential conflicts of interest.
REFERENCES
1. Wolff AC, Hammond MEH, 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 2. 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. Arch Pathol Lab Med 131:18, 2007 [Epub ahead of print][Medline] 3. Vincent-Salomon A, MacGrogan G, Couturier J, et al: Calibration of immunohistochemistry for assessment of HER2 in breast cancer: Results of the French multicentre GEFPICS study. Histopathology 42:337-347, 2003[CrossRef][Medline] 4. Mass R: The role of HER-2 expression in predicting response to therapy in breast cancer. Semin Oncol 27:46-52; discussion 92-100, 2000[Medline] 5. Tubbs RR, Pettay JD, Roche PC, et al: Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: Apparent immunohistochemical false-positives do not get the message. J Clin Oncol 19:2714-2721, 2001 6. 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 7. Romond EH, Perez EA, Bryant J, et al: Trastuzumab plus Adjuvant Chemotherapy for Operable HER2-Positive Breast Cancer. N Engl J Med 353:1673-1684, 2005 8. Smith I, Procter M, Gelber RD, et al: 2-year follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer: A randomised controlled trial. Lancet 369:29-36, 2007[CrossRef][Medline] 9. Herceptin Package Insert (November 2006). Genentech Inc, South San Francisco, CA, 2006. http://www.gene.com/gene/products/information/oncology/herceptin/insert.jsp
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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2.0 were eligible for the trastuzumab adjuvant trials.
