Affiliations of authors: P. C. Roche, V. J. Suman, R. B. Jenkins, J. N. Ingle, Mayo Clinic and Mayo Foundation, Rochester, MN; N. E. Davidson, Eastern Cooperative Oncology Group Data Management Office, Brookline, MA; S. Martino, Southwest Oncology Group Operations Office, San Antonio, TX; P. A. Kaufman, Cancer and Leukemia Group B Data Management Center, Durham, NC; F. K. Addo, Medcenter One Health Systems, and Mid Dakota Clinic, Bismarck, ND; B. Murphy, Metro-Minnesota Community Oncology Program, St. Louis Park, MN; E. A. Perez, Mayo Clinic and Mayo Foundation, Jacksonville, FL.
Correspondence to: Edith A. Perez, M.D., Mayo Clinic, Division of Hematology/Oncology, 4500 San Pablo Rd., Jacksonville, FL 32224 (e-mail: perez.edith{at}mayo.edu).
ABSTRACT
The efficacy of trastuzumab for metastases coupled with the relatively poor prognosis of patients with node-positive, HER2-positive breast cancer has led to the evaluation of trastuzumab as an adjuvant therapy. A prospective, randomized, three-arm, phase III trial is being conducted by the Breast Intergroup (N9831) for women with primary, operable, histologically confirmed, node-positive breast carcinoma that strongly overexpresses (3+) HER2 protein and/or displays HER2/neu gene amplification, as determined by local laboratory testing. The protocol requires confirmatory central testing of HER2 status using the HercepTestTM immunohistochemistry and the Vysis PathVysionTM fluorescence in situ hybridization (FISH) assays. Tumor specimens from the first 119 patients enrolled in N9831 were centrally tested; 74% were found to be HercepTestTM 3+ and 66% were found to have HER2 gene amplification. Only six of nine (67%) of the specimens submitted by local laboratories as FISH positive could be confirmed by central assays. The concordance for central HercepTestTM and central FISH assays was 92%. The poor concordance (74%) between local and central testing for HER2 status has led to modifications in the eligibility criteria for N9831.
One of four ongoing large-scale trials evaluating adjuvant trastuzumab is N9831, a prospective, randomized, three-arm, phase III trial conducted by all members of the Breast Intergroup (Cancer and Leukemia Group B, Eastern Cooperative Oncology Group, Southwest Oncology Group, and North Central Cancer Treatment Group [NCCTG]), with the NCCTG as the coordinating center (trial N9831; http://www.cancer.gov/clinical_trials/finding/) (3). Women with primary operable, histologically confirmed, node-positive breast adenocarcinoma that strongly overexpresses HER2 protein or displays HER2/neu gene amplification in local laboratories are eligible for the trial. Measurement of HER2 protein may be performed by the Food and Drug Administration (FDA)-approved DAKO HercepTestTM (package insert 2001; DAKO, Carpinteria, CA) (3+ score) or another immunohistochemical assay where one third or more invasive tumor cells stain positive (3+) with an anti-HER2 antibody. Gene amplification assays using fluorescence in situ hybridization (FISH) may be performed by PathVysionTM (package insert 2001; Vysis, Inc., Downers Grove, IL) (2.0 HER2/neu probe/control probe ratio considered amplified) or INFORM (package insert 2001; Ventana Medical Systems, Inc., Tucson, AZ) (
5 gene copies of HER2/neu considered amplified). Women whose tumors stain 02+ by local community-based immunohistochemistry (IHC) but demonstrate amplification by local community-based FISH are eligible for the study. A schematic showing the randomization and treatment regimen can be found at the Journal's Web site (supplemental Fig. 1, http://jncicancerspectrum.oupjournals.org/jnci/content/vol94/issue11/index.shtml). A secondary endpoint of N9831 is to assess the concordance between HER2 protein overexpression and HER2/neu gene amplification as determined by HercepTestTM and FISH (using PathVysionTM), respectively, at a central testing facility.
We report the results of a planned analysis of the first 119 tumor specimens that underwent central HER2 testing, which assesses the level of agreement between local community HER2 testing and central HER2 testing and the concordance between central HER2 HercepTestTM and central HER2/neu FISH results. The N9831 protocol indicated that if a discrepancy of more than 20% was observed between local and central testing for HER2 status in this analysis, then consideration would be given to changing the provision that patient eligibility be based solely on local HER2 results.
Sections from 119 paraffin-embedded breast tumor specimens were analyzed for HER2 overexpression using the HercepTestTM and a DAKO Autostainer in strict accordance with the manufacturer's instructions in the Immunohistochemistry Laboratory (P. C. Roche, Mayo Clinic, Rochester, MN). The stained slides were interpreted and scored on a 03+ scale according to the FDA-approved guidelines for HercepTestTM by the study pathologist. FISH (protocol available at the Journal's Web site [supplemental protocol http://jncicancerspectrum.oupjournals.org/jnci/content/vol94/issue11/index.shtml]), is performed with the PathVysionTM HER2/neu DNA probe kit following the manufacturer's instructions in the Cytogenetics Laboratory (R. B. Jenkins, Mayo Clinic, Rochester, MN). An area of the invasive carcinoma is identified from a hematoxylin-and-eosin-stained serial section from each specimen and outlined on the test slide. Two technologists independently count and analyze the number of fluorescent signals in each of 30 tumor cell nuclei as described (4).
As of July 15, 2001, the specimens of 119 women enrolled on N9831 have been centrally evaluated for HER2 status (Table 1). HER2 status at enrollment was determined in one of 65 different local U.S. laboratories by the HercepTestTM (50%), other IHC methods (42%), Vysis FISH (7%), or Ventana FISH (1%). Only two of these 65 local laboratories were used by more than 5% of the women.
|
|
The poor concordance (74%) between local laboratory results of HER2 positivity and central laboratory findings using HercepTestTM or FISH is of great concern because the benefit of trastuzumab, at least in retrospective studies, appears to be observed primarily in patients whose tumors overexpress HER2 protein by IHC or gene amplification by FISH (57). So far, our results indicate that as many as 26% of the patients already enrolled using local testing for entry criteria may not be the best candidates to determine whether anti-HER2 monoclonal antibody therapy is advantageous as an adjuvant for chemotherapy. We did not address whether the volume of HER2 tests performed by local laboratories influenced the concordance with our central testing or whether a particular testing method may be contributing to the discordance. Only two of 65 laboratories were used by more than 5% of the women. Thus, our data do not allow us to conclude whether testing only in other reference laboratories or changes in methodology would eliminate the problem of discordance.
We have chosen to modify the HER2 testing requirement for our N9831 clinical trial 1) to ensure the enrollment of patients with the highest probability of benefiting from anti-HER2 monoclonal antibody therapy [such patients would be the most appropriate population to address the primary aim of this clinical trial (trial N9831; http://www.cancer.gov/clinical_trials/finding/) (3)], and 2) to ensure the protection of patients who do not strongly overexpress HER2 from the experimental aspects of treatment with trastuzumab. A notification regarding discordant findings of HER2 testing has been sent to physicians and patients already participating in this clinical trial. Protocol eligibility was modified so that a woman can enroll onto the trial if she has node-positive breast cancer that is found to strongly overexpress HER2 or has HER2/neu gene amplification by central testing or by a local laboratory. If the woman was enrolled on the basis of local laboratory testing, then central confirmatory testing will be performed before the completion of the first chemotherapy regimen. After central review, if the tumor specimen is found to strongly overexpress HER2 (3+ positivity by HercepTestTM) or has HER2/neu amplification by FISH, then the patient will continue protocol treatment as randomly assigned. However, if the tumor specimen does not meet the central testing criteria, the specimen will be sent to an additional reference laboratory for review and, if the testing is still negative, then the patient and her physician will be informed of the findings, and adjuvant therapy will be continued at the individual physician's discretion.
In summary, the overall performance of HER2 testing by local laboratories in this initial sample of 119 patients was disappointing, with unacceptably high levels of discordance with central testing. Although most of our experience involved IHC, finding three discordant cases of nine tested by FISH indicates the need for further evaluation of this assay as used by local oncology and pathology practices. Specific guidelines to gain proficiency in HER2 testing are difficult to specify, but there is general agreement that this is related to the volume of testing and rigorous quality-control measures, including correlative studies involving both IHC and FISH. Based on the findings with this initial sample and the critical importance of accurate HER2 testing for optimal patient care, we conclude that further study of the inter-laboratory agreement is indicated.
NOTES
Note added in proof: Specimens from 370 women were centrally tested as of February 21, 2002. The results are nearly identical to those presented in this report for 119 women. Seventy-five percent (279/370) of enrolled women had tumors that are 3+ by central IHC, and 71% (263/370) had tumors that are amplified by central FISH. Of 36 women (10%) enrolled with local FISH testing, 23 (64%) had tumors that are amplified by central FISH and 26 (72%) had tumors that are 3+ by central IHC. The concordance for central IHC and central FISH is 92% (342/370).
R. B. Jenkins serves on the Scientific Advisory Board of Vysis, Inc., and conducts research sponsored by the company.
P. A. Kaufman conducts research sponsored by Genentech, and is a member of the Genentech Speaker's Bureau.
E. A. Perez conducts research sponsored by Genentech.
Genentech is providing a research grant to partially cover the data analysis (including HER2 testing and cardiac testing) in this trial.
This study was conducted as a collaborative trial of the North Central Cancer Treatment Group and Mayo Clinic and was supported in part by Public Health Service grants CA25224, CA37404, CA15083, CA63826, CA52352, CA63849, CA35195, CA35272, CA60276, CA35269, CA37417, CA63848, CA35101, CA35415, CA35103, and U10CA2522421 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Other support for this study was provided by grants from the Breast Cancer Research Foundation and Genentech.
REFERENCES
1
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:78392.
2
Cobleigh MA, Vogel CL, Tripathy D, Robert NJ, Scholl S, Fehrenbacher L, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:263948.
3 Perez EA, Comis RL, Kaufman PA, Martino S. Phase III randomized study of doxorubicin plus cyclophosphamide followed by paclitaxel with or without trastuzumab (Herceptin) in patients with HER-2-overexpressing breast cancer. [Accessed 04/30/02.] Available from: http://www.cancer.gov/clinical_trials/finding/.
4 Perez EA, Roche PC, Jenkins RB, Reynolds CA, Halling KC, Ingle JN, 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 2002;77:14854.[Medline]
5 Mass RD, Sanders C, Charlene K, Johnson L, Everett T, Anderson S. The concordance between the clinical trials assay (CTA) and fluorescence in situ hybridization (FISH) in the Herceptin pivotal trials [abstract 291]. Proc ASCO 2000;19:75a.
6 Mass RD, Press M, Anderson S, Murphy M, Slamon D. Improved survival benefit from Herceptin (trastuzumab) in patients selected by fluorescence in situ hybridization (FISH) [abstract 85]. Proc ASCO 2001;20:22a.
7 Vogel CL, Cobleigh M, Tripathy D, Mass R, Murphy M, Stewart SJ, et al. Superior outcomes with Herceptin (trastuzumab) (H) in fluorescence in situ hybridization (FISH)-selected patients [abstract 86]. Proc ASCO 2001;20:22a.
Manuscript received November 29, 2001; revised April 2, 2002; accepted April 23, 2002.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |