HER2 testing: state of the laboratories.
Article Type: Editorial
Subject: Breast cancer (Genetic aspects)
Breast cancer (Diagnosis)
Breast cancer (Care and treatment)
Genetic screening (Usage)
Author: Mohsin, Syed K.
Pub Date: 05/01/2010
Publication: Name: Archives of Pathology & Laboratory Medicine Publisher: College of American Pathologists Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 College of American Pathologists ISSN: 1543-2165
Issue: Date: May, 2010 Source Volume: 134 Source Issue: 5
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 230246551
Full Text: It has been more than 2 decades since the first publication describing the potential role of HER2 in breast cancer. A variety of techniques were employed in the initial studies to assess HER2 at DNA, RNA, and protein levels. These studies, mostly retrospective, demonstrated that HER2 is a marker of poor prognosis and suggested that it may be a predictive marker of response to certain types of chemotherapy. Like hundreds of other biomarkers of potential significance in breast cancer, interest in the clinical use of HER2 remained subdued. The development of a targeted treatment to HER2 and indirectly this pathway (ie, Herceptin [trastuzumab], Genentech, Inc, South San Francisco, California) changed the course of history for HER2. As soon as the clinical trials for Herceptin began, initially in patients with metastatic breast cancer and later in an adjuvant setting, HER2 became a clinically useful biomarker, (1) and it met 1 of the 3 pivotal requirements for biomarker validation, as described by McGuire. (2)

The journey to validate HER2 assays has faced several obstacles during nearly one and one-half decades. It has certainly lagged significantly behind estrogen and progesterone receptor assay validation. One would expect that the experiences gained from hormone receptor assay validation would have helped, but that does not seem to be the case. It is fair to say that, at this time, there is no true gold standard for HER2 testing in breast cancer, and if guidelines published by the National Institutes of Health and McGuire (2) are considered, not a single study exists that fully addresses the critical steps needed for such a validation study. These guidelines require 3 important steps: technical validation, clinical validation, and clinical usefulness.

The main difficulty in developing a clinically useful HER2 test has been the relative lack of, or access to, clinically well-defined sets of breast cancer samples, against which either one or more tests could be directly tested and validated. Therefore, most of the efforts have been focused on the technical validation of primarily immunohistochemical assays, often comparing them to the gene amplification status by florescent in situ hybridization (FISH), serving as an intermediate standard to compare with other assays. However, most of the reports, either in community practice or in evaluations in clinical trial settings, have fallen short of the expectations. (3-7)

See also p 728.

The initial steps for HER2 testing focused on developing good antibodies against HER2. These antibodies varied tremendously (12% to 42%) in their sensitivity to detect HER2 overexpression in breast cancer. The issue of specificity is difficult to answer because a specific target for clinical outcome was not clearly defined in the initial stages, even though predictions about the response to Herceptin became the driving force for HER2 testing. For FISH assay, relatively few reagents were developed, and 2 assays received US Food and Drug Administration (FDA) approval. It was proposed that HER2 gene amplification become the standard to which other assays need to be validated. This was partly based on results from the initial clinical trials, which showed better response rates to HER2 gene-amplified breast cancers. Therefore in our current understanding, the technical validation of HER2, in general, often refers to concordance with a FISH assay. According to guidelines for assay validation, all nonvalidated assays must be compared with one that has been thoroughly validated clinically in actual patient samples treated in a uniform fashion, ideally in a randomized clinical trial. Per the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines, a 95% concordance to a previously "validated" assay is sufficient. However, 95% concordance to another assay, which was validated using the same rules, can potentially take the new assay far away from the actual validation point. Thus, there appears to be some differences in how some guidelines define steps and requirements for validation studies.

It is also important to point out some of the unique features that were encountered in studies that attempted to validate HER2 assays. First, an unusual scoring system of 0, 1+, 2+, and 3+ was employed. For the scoring of almost all other biomarkers, pathologists have used a combination of percentage or proportion of positive cells with intensity of staining (eg, H-score, Allred score). In general, pathologists use these simple scoring algorithms in routine practice and feel comfortable with them. Second, FDA approval was sought early in the process of validation, when clinically well-defined tissue samples were scarce at best. Most experts agree that such approval did not meet scientific guidelines and led to a great deal of confusion among laboratories deciding to perform the HER2 assay in-house. Third, the initial guidelines from ASCO/CAP did not put much emphasis on the preanalytic variables that affect these assays. The current guidelines addressed those shortcomings and emphasis was placed on the requirements for tissue fixation. (8) These guidelines are a giant step forward in the arena of prognostic and predictive markers and reflect the increasing collaboration between the pathologists and oncologists in daily practice. With this background of difficulties in HER2 testing and almost 2 years after publication of the most recent guidelines, let us look at what Nakhleh et al (9) have reported in their survey.

As pointed out by Nakhleh et al, (9) 2 very positive aspects are obvious from their data. First, it seems that most laboratories have embraced the ASCO/CAP guidelines as shown by the number of laboratories trying to improve the technical quality of their HER2 assay using the guidelines. However, it is not possible to understand the magnitude of that improvement because of a lack of similar prior survey data. Second, more than 90% of the laboratories report having a policy and program for assessing the competency of pathologists who interpret and report HER2 results in their laboratories.

About 60% of laboratories reported that they compared/ validate their in-house HER2 assay with another "validated" assay. Data obtained from the CAP HER2 competency survey (9) show that the laboratories use more than 7 different antibodies and assays. A critical look at the data from these laboratories show that one-half the laboratories are using less than 40 cases for their comparison studies with either FISH or another immunohistochemistry (IHC) assay in another laboratory. The CAP guidelines for validation suggest between 25 and 100 samples for validation. Some other studies suggest that, for an HER2 assay, a minimum of 40, and ideally about 60 to 80 cases or samples, are needed for reasonable data comparison. Looking at the data comparing IHC to FISH, 81% and 73% of laboratories reached the 95% concordance level for negative and positive test results, respectively. If we combine the numbers mentioned above, it seems that, for truly positive HER2 cases, only about 25% of the laboratories have used the required number of cases for a reliable correlation study with FISH that reaches 95% concordance. Because a significant number of laboratories also used 2+ IHC cases, which is an indeterminate result and ideally should not be used for correlation studies, then this percentage falls even lower. The validation studies comparing one IHC assay to another IHC assay in another laboratory show even less-convincing data.

The other important aspect of the ASCO/CAP recommendations was their emphasis on the preanalytic variable of tissue fixation. The recommendations were made to consider a minimum of 6 hours and not more than 48 hours of fixation in a 10% neutral-buffered formalin. From the data reported by Nakhleh et al, (9) only 27% of the laboratories report the exact fixation time in their reports. Some laboratories have a general statement that they meet the published guidelines. Therefore, it is reasonable to assume that despite efforts to meet these guidelines for fixation times, most laboratories, at this time, are unable to extract and report the actual time of tissue fixation for their samples. The other data elements in this report regarding tissue fixation suggest that most laboratories are trying to understand the importance of this preanalytic variable and making efforts to address it. It will be interesting to see whether there is additional improvement in these numbers in a follow-up study.

There are several aspects of improvement in HER2 testing. These can be categorized in 3 areas: technical aspects, gaps in guidelines and recommendations, and multiplex testing algorithms. For the technical aspects, the sensitivity of all HER2 assays needs to be established in reliable tissue samples. Because fixation is an important aspect, cells lines with controlled formalin fixation and known HER2 membrane receptor densities, or at least a small subset of patient samples (200-300) with known ischemic and fixation times, should be used. The next step is to define a cutoff for a positive test result. That step generally requires starting with a reasonable spread of signals, that is, the recording protein expression as a continuous variable. Thus, the H-score or Allred scoring systemprovides both reproducible and easy-to-use systems. Using such a strategy is more likely to provide a clinically meaningful cutoff for IHC assays for HER2, similar to what was obtained for hormone receptors. The predictive value of such a test then needs to be verified in a randomized clinical trial. As some of the clinical trials on Herceptin (such as NSABP B-31, BCIRG 006, NCCTG 9831) mature, some of these pivotal validation studies can be completed.

The second area for improvement is in the details provided in the guidelines for HER2 assays. These recommendations need to be reasonable to accommodate a variety of clinical practice settings and should include practical ways to address issues regarding ischemic times, total length of tissue fixation, and more details about comparison and validation studies. Because the tissue-fixation time starts either in a radiology suite or in an operating room, radiologists and surgeons should be involved in this collaborative effort. It has started to become clear that preanalytic variables in predictive marker testing are critical steps. The idea that such tests be limited to certain reference laboratories may not offer a true solution if the tissue is not adequately preserved, and that step is going to occur in the community and academic pathology practices. In the guidelines for validations, a general rule of testing 25-100 samples, without much reference to the specific needs for HER2 validation, may not be sufficient. In addition, CAP and ASCO should consider working more closely with the FDA to ensure that FDA approval of future HER2 assays meet the scientific guidelines.

Finally, it is worth asking whether we have reached the ceiling for 95% concordance using a single marker or assay type to select patients for a costly treatment like Herceptin. As the trend toward cost containment in health care continues, pressure for a nearly perfect predictive marker for Herceptin will rise. In other areas of breast cancer care, tests based on multiple genes seem to provide more consistent prognostic information (eg, Oncotype DX [Genomic Health, Inc, Redwood City, California], MammaPrint [Agendia, Amsterdam, the Netherlands]). It may be possible to develop tests based on multiple relevant genes or proteins to better predict the response to Herceptin.


(1.) Cobleigh MA, Vogel CL, Tripathy D, 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(9):2639-2648.

(2.) McGuire WL. Breast cancer prognostic factors: evaluation guidelines. J Natl Cancer Inst. 1991;83(3):154-155.

(3.) Paik S, Bryant J, Tan-Chiu E, et al. Real-world performance of HER2 testing: National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst. 2002;94(11):852-854.

(4.) 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. 2002;94(11):855-857.

(5.) Reddy JC, Reimann JD, Anderson SM, Klein PM. Concordance between central and local laboratory HER2 testing from a community-based clinical study. Clin Breast Cancer. 2006;7(2):153-157.

(6.) O'Malley FP, Thomson T, Julian J, et al. HER2 testing in a population-based study of patients with metastatic breast cancer treated with trastuzumab. Arch Pathol Lab Med. 2008;132(1):61-65.

(7.) 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. 2007;131(1):18-43.

(8.) Dowsett M, Hanna WM, Kockx M, et al. Standardization of HER2 testing: results of an international proficiency-testing ring study. Mod Pathol. 2007;20(5): 584-591.

(9.) Nakhleh RE, Grimm EE, Idowu MO, Souers RJ, Fitzgibbons PL. Laboratory Compliance with the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) Guidelines for Human Epidermal Growth Factor Receptor 2 (HER2) testing: A College of American Pathologists Survey of 757 Laboratories. Arch Pathol Lab Med. 2010;134(5):728-734.

Syed K. Mohsin, MD

Accepted for publication January 15, 2010.

From the RMH Pathology Associates, Riverside Methodist Hospital, Columbus, Ohio.

Reprints: Syed K. Mohsin, MD, RMH Pathology Associates, Riverside Methodist Hospital, 3535 Olentangy River Rd, Columbus, OH 43214 (e-mail: smohsin@ohiohealth.com).
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