What is the optimal algorithm for assessment of tumor HER2 status?

OVERVIEW: Approximately 20-30% of women with breast cancer express HER2 gene amplification, which along with protein overexpression are strong prognostic factors and predictors of response to trastuzumab. HER2 status determination is an important component in the diagnostic evaluation of a patient with breast cancer. The various methods for determining HER2 status measure gene amplification, RNA overexpression, or protein overexpression. HER2 protein overexpression is frequently measured by immunohistochemistry (IHC), while fluorescence in situ hybridization (FISH) detects HER2 gene amplification. Results obtained with IHC and FISH are not always concordant. The clinical outcomes associated with trastuzumab are dependent upon the HER2 status of the patient. Although patients with 2+ HER2 protein overexpression do experience benefits, the response rates for trastuzumab, as a single agent and in combination with chemotherapy, are greater for patients with 3+ HER2 protein overexpression. Retrospective analyses demonstrate that patients with HER2 gene amplification are more likely to benefit from trastuzumab with respect to overall response and survival. Since the scientific literature on the subject of HER2 is still evolving, clinicians are advised to follow this topic closely as it continues to develop.

	In approximately what percentage of your patients with primary breast cancer do you assess HER2 status?	100%
	In the last year, approximately how many women have you evaluated and/or treated with metastatic breast cancer?	Median = 53
	In approximately what percentage of your patients with metastatic breast cancer do you attempt to assess HER2 status?	100%
	About how many patients were HER2-positive?	Median = 10

How often do you obtain FISH results on a patient you are treating?

DISCORDANCE IN THE ASSESSMENT OF HER2 STATUS

There is a significant problem with HER2 testing in the community. When we designed our adjuvant study — evaluating trastuzumab in combination with chemotherapy — we included a plan for central analysis of HER2 status. Unfortunately, when we looked at the initial 119 patients, we found a high level of discordance in HER2 testing by IHC, and even by FISH, when comparing measurements in the community versus central testing.

We found discordant results in six of the nine FISH test assays. These were FISH-positive in the community, but FISH-negative in the central lab. The number of patients is very, very small to date, so we cannot conclude that FISH is a bad test to be performed in the community, but we need to look into why this discordance occurs. IHC concordance between the community and central laboratories was about 75 percent.

We've done another study of HER2 testing, based on 1,500 specimens sent to Mayo medical laboratories over a five-month period. We took 213 specimens labeled as HER2 2+ and evaluated them for protein overexpression and gene amplification, and we found that only 12 percent of the tumors scored as 2+ by the HercepTest™ actually were FISH-positive.

—Edith A Perez, MD

ASSESSING HER2 STATUS

The NCCN guidelines call for HER2 testing of all breast cancers; however, this year we were much more specific than in the past. We call for HER2 testing by IHC, and if the IHC result is 2+ by the HercepTest™, we call for FISH analysis. This is primarily because in the metastatic setting, when you're looking for benefit or lack thereof from trastuzumab, FISH-positivity is by far the best predictor of responsiveness.

Women whose breast cancers are IHC 3+ by the HercepTest™ are almost all FISH-positive, while those that are IHC 0 or 1+ are almost always FISH-negative. This approach is based upon the study reported by Chuck Vogel, which looked at trastuzumab as a single agent and found very good rates and long duration of response in those women who were either IHC 3+ or IHC 2+ and FISH-positive.

—Robert W Carlson, MD

HER2 ASSESSMENT

Reliable detection of HER2 overexpression is important for the success of trastuzumab (Herceptin®) therapy. Several methods are available for measuring HER2 expression at the DNA, RNA or protein level. The method most frequently employed is immunohistochemical (IHC) detection of the HER2 receptor in paraffin sections. Advantages include the precise localization of the HER2 protein, the availability of paraffin material and the ease of the procedure.

However, IHC can be influenced by the sensitivity/specificity of the antibody, tissue treatment and, in particular, subjective assessment. These disadvantages do not exist in the detection of gene amplification by fluorescence in situ hybridization (FISH) or polymerase chain reaction. However, FISH requires expensive equipment that is not widely available in pathology laboratories. Another approach quantitates shed HER2 antigen in the serum by an enzyme-linked immunosorbent assay.

The key advantage of this method is the ease of sampling blood, however, serum HER2 concentrations do not accurately reflect the tumor status. Furthermore, this method does not register single-cell expression, which is important for therapeutic decision-making. For routine diagnostics, the combination of IHC and FISH is useful. In addition, to improving the accuracy and comparability of HER2 assays, these optimized protocols may further enhance the efficacy of t rastuzumab thera py by selecting those patients most likely to respond.

—Gerhard Schaller, MD et al.
Ann Oncol 2001;(Suppl 1):S97-S100.

HER2 REPRODUCIBILITY

Consensus regarding the best methods, reagents, or cut-off points to define HER2 status for determining trastuzumab responsivity has not yet been reached. HER2 testing for other prognostic or predictive purposes, e.g. to determine whether patients are likely to respond to other agents, such as dose-intensive doxorubicin, may be less. Data from the Cancer and Leukemia Group B trial 8541 (companion 8869) suggest that, with proper controls in high-volume laboratories, many of the available methods produce comparable results.

—Ann Thor, MD
Ann Oncol 2001;(Suppl 1):S101-S107.

Molecular targets for determining HER2 status and the tests used to detect these molecules. (mRNA = messenger RNA, ECD = extracellular domain, ELISA = enzyme-linked immunosorbent assay, IHC = immunohistochemistry, CISH = chromogenic in situ hybridization, PCR = polymerase chain reaction, and FISH = fluorescence in situ hybridization)

Adapted from Schaller et al. Ann Oncol 2001;12:(Suppl 1):S97-S100

* = calculated, ACIS™= automated cellular imaging system, CTA= clinical trial assay (4D5 and CB11 antibodies)

Frequency of IHC scores according to level of HER2 gene amplification.
(Pauletti G et al. J Clin Oncol 2000 ; 18:3651-3664)

Level of HER-2 Gene Amplification (HER2/neu signals per cell)

Response rates for weekly trastuzumab and paclitaxel according to HER2 expression . (Seidman AD et al. J Clin Oncol 2001; 19:2587-2595)

HER2 Status

Percent of Patients with HER2 Gene Amplification According to Immunohistochemistry Score (IHC)

CTA = clinical trial assay (4D5 and CB11 antibodies)

Overall response rates for trastuzumab trials in breast cancer according to IHC score. (t=trastuzumab, A=anthracycline, C=cyclophosphamide)

1. Cobleigh MA et al. J Clin Oncol 1999;17:2639-2648. Abstract
2. Herceptin® [Package Insert], 2000.
3. Uber KA et al. , Pro ASCO 2001; Abstract 1949.
4. Burstein HJ et al. J Clin Oncol 2001;19:2722-2730. Abstract

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