EDITORIAL

Are Patterns of HER-2/neu Amplification and Expression Among Primary Tumors and Regional Metastases Indicative of Those in Distant Metastases and Predictive of Herceptin Response?

Ann Thor

Correspondence to: Ann Thor, M.D., University of Oklahoma College of Medicine, 940 Stanton L. Young Blvd., BMSB 451, Oklahoma City, OK 73104.

HER-2/neu (ERB-B2), a member of the receptor tyrosine kinase superfamily, is altered by gene amplification and/or protein overexpression in a wide variety of human epithelial malignancies. Such alterations activate signaling systems that promote cell growth, angiogenesis, cancer metastases, and other procarcinogenic pathways (1). These HER-2 abnormalities are found in approximately one third of breast cancers. HER-2 "status" is a loose and suboptimal term that is commonly used to describe the degree of HER-2 gene amplification and/or the extent of Her-2 protein overexpression. HER-2 status has been used as a marker for breast cancer prognosis (in addition to the classic markers lymph node status, tumor size, type and grade, hormone receptor status, and proliferation rate), because, in the majority of cases, HER-2 alterations independently portend a worse outcome. In addition, associations have been found between the degree of HER-2 expression or gene copy number and the likelihood that a patient may derive benefit from some chemotherapy regimens, i.e., predictive value (24). Statistically significant interactions between HER-2 status and chemotherapeutic agents other than anthracyclines have also been reported (5). Given these chemotherapeutic/HER-2 interactions and new anti-HER-2 therapeutic agents, it is no longer always correct to assume that cancers with an altered HER-2 status will have a poor outcome. As noted, HER-2 has become an important target for novel therapeutic strategies, such as trastuzumab (Herceptin), a monoclonal antibody that acts as an HER-2 antagonist. This agent has shown promise in treating a subset of patients with otherwise chemorefractory, late-stage breast cancer. Many recently opened breast cancer trials combine Herceptin with other agents to treat various stages of disease.

There are several laboratory methods to evaluate HER-2. These include fluorescent in situ hybridization (FISH), to determine HER-2 gene copy number, reverse transcription–polymerase chain reaction, to measure HER-2 messenger RNA levels, and immunohistochemical (IHC) analysis, to detect overexpressed HER-2. Any or all of these methods can be used to determine the HER-2 status of a given tumor. The majority of these methods use a single sample of formalin-fixed tissue from the primary breast cancer. However, it remains unclear which method, reagent, or protocol provides superior prognostic or predictive data. None of these methods determine whether the effectors downstream of HER-2 are activated, whether HER-2 is bound to the EGF receptor (erbB-1) or to its other dimerization partners, whether HER-2 is phosphorylated and thus in its activated configuration, or whether HER-2 is internalized or recycled to the membrane. Assays that could delineate these important and biologically relevant variables or pathways, or a single sentinel test to better assess the functional status of the receptor, need to be developed.

The study by Simon et al. (6) in this issue of the Journal utilizes both IHC and FISH to determine the HER-2 status of primary breast tumors and regional (axillary) metastases. The study was designed to compare HER-2 data derived by each method as well as the HER status of primary breast cancers and their associated metastases. The reader should be clear that, in this study, the metastases being analyzed are regional, concurrent lymph node metastases from the primary cancer rather than distant (usually visceral or bony) metastases. One should not assume that the two types of metastases are equivalent, since distant metastases often represent clonal outgrowths with genetic and other modifications that are often identified years after resection of the primary cancer. Furthermore, cells that metastasize locally via lymphatics may be biologically distinct from those that have the capacity to invade the vasculature, travel to distant sites, and establish clinically significant disease. That Simon et al. ( 6 ) have detected discordance in HER-2 status between the primary tumor and involved axillary lymph node metastases in about 5% of the pairs tested is important, but this small difference in HER-2 status may represent only the proverbial "tip of the iceberg." We have found a nearly 20% discordance in HER-2 expression between primary tumors and distant, visceral metastases utilizing either IHC or FISH [ (7); Edgerton SE, Merkel D, Moore DH, Thor AD: unpublished data].

The study by Simon et al. (6) confirms a strong and statistically significant relationship between HER-2 data generated by either FISH or IHC. For example, among the 597 tumor samples that had a HercepTest score of 2+ or 3+, 97% and 99%, respectively, displayed HER-2 gene amplification by FISH, while 3.5% of the 2115 tumor samples with a HercepTest score of 0 showed HER-2 gene amplification by FISH. The authors reported that 66% of the 145 tumors that had a HercepTest score of 1+ had amplified HER-2, which is a higher percentage than that reported by others (8, 9). Low-level discordance between IHC and FISH data among tumors that have HercepTest scores of 0 or 1+ and variable levels of gene amplification in tumors that have a HercepTest score of 2+ have been used as evidence by some (810) to support a recommendation that FISH should be performed in all, or perhaps a specific subset of, immunohistochemically defined tumors. Randomized studies comparing the results of IHC and FISH assays to determine HER-2 status with outcome/response data from patients treated with HER-2 antagonists will be useful for both cost/benefit analyses and to determine which method, if either, is superior for predicting treatment response.

Some issues about how these tests for HER-2, especially FISH, were applied in this study by Simon et al. are worthy of discussion (6). For example, the authors used modifications of the manufacturer's recommended method to score HER-2 amplification and a higher cut point than that recommended by the manufacturer for the HER-2-to-centromere 17 signal ratio to analyze their data (PathVysionTM HER-2 DNA Probe Kit Package Insert, Vysis Inc., Downers Grove, IL). Moreover, the authors used 4'6-diamidino-2-phenyolindole dihydrochloride (DAPI), hydrate, fluorescein isothiocyanate, and Texas Red microscope filters to detect immunofluorescent signals for DNA, HER-2, and centromere 17, respectively, rather than the DAPI Green dual- or triple-bypass filters and DAPI/9 Orange dual-bypass filters recommended by the manufacturer (Vysis Inc.). This choice of microscope filters may explain why the authors report weak and uninterpretable immunofluorescent signals in some cases. In our experience, the recommended filters provide enhanced signal detection for the SpectrumOrangeTM-labeled HER-2 probe. While these technical issues are relatively minor, the reader should be aware that FISH methodologies for the detection of HER-2 gene amplification may vary (there are two different manufacturers of reagent kits), and laboratories that make changes in either the recommended equipment or methodology may compromise the performance of either test. In this regard, future goals of the pathology community should include efforts to reduce variance in laboratory methodology, utilization of standards and comparable controls, disclosure of the methods utilized when deviations from commonly accepted protocols are made [as appropriately done by Simon et al. (6) ], written disclosure of reagent sources, and adoption of uniform standards of reporting for each tumor specimen, as recommended by the guidelines published by the College of American Pathologists (10).

In summary, the article by Simon et al. (6) contributes to our understanding of HER-2 testing issues as they relate to breast cancer treatment response. The authors' conclusion, that there is some variance in HER-2 status between primary and regionally metastatic disease, is supported by their data. In 5% of patients, an HER-2-negative primary breast tumor was associated with an HER-2-positive or partially HER-2-positive regional metastasis; occasionally, the reverse has been observed. Given these results, should we consider testing the HER-2 status of metastatic lesions if they are available for biopsy before Herceptin treatment? This investigator suggests that additional data derived from studies of nonregional metastases or stage progressed disease should be evaluated for clinical relevance before such a policy is globally adopted. However, for individual patients for whom no other treatment options are available or for whom treatment with HER-2 antagonists is sought, it may be clinically appropriate to use FISH methodology to retest metastatic disease. For the majority of samples tested by Simon et al. (6), the level of variance between results obtained with FISH and IHC was low, and differences in test performance are not likely to explain why some patients failed to respond to Herceptin immunotherapy. Other issues, such as HER-2 heterodimerization partners and receptor activation of downstream signaling pathways, may be of greater clinical relevance to Herceptin responsiveness. Adherence to uniform standards (10,11) for IHC and FISH techniques may facilitate laboratory testing of HER-2 status and allow data to be compared such that eventually these issues may be resolved.

REFERENCES

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