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Analytic Uncertainty and Clinical Impact of Her2/neu Testing

Surgical Rounds®, July 2006, Volume 0, Issue 0

Richard M. Peterson, Chief Surgical Resident, Department of Surgery, St. Agnes Hospital, Baltimore, MD Armando Sardi, Director of the Institute for Cancer Care, Chief of Surgical Oncology, Mercy Medical Center, Baltimore, MD Michael Ballo, Co-Director of Cytopathology, Department of Pathology, St. Agnes Hospital, Baltimore, MD

Richard M. Peterson, MD, MPH Chief Surgical Resident

Department of Surgery

St. Agnes Hospital

Baltimore, MD Armando Sardi, MD Director of the Institute for Cancer Care

Chief of Surgical Oncology

Mercy Medical Center

Baltimore, MD Michael Ballo, MD Co-Director of Cytopathology

Department of Pathology

St. Agnes Hospital

Baltimore, MD

Human epidermal growth factor receptor–2 (HER2/neu) is a cell surface receptor with tyrosine kinase activity that aids in cell growth regulation and differentiation. Overexpression of HER2/neu protein occurs in various malignant neoplasms, including those arising in the breast, and correlates with certain clinical and histologic features. Overexpression in those with metastatic disease suggests there will be a poorer response to nonspecific anticancer therapies and that these patients would be more effectively treated with trastuzumab (Herceptin®). Recently developed laboratory tests for HER2/neu are more sensitive and specific and should be used to determine whether a patient is a good candidate for targeted therapy with trastuzumab.


It has become increasingly important to identify factors associated with breast cancer that impact prognosis and influence treatment strategies. Breast cancer is the most common malignancy in women and constitutes 32% of new cancer cases.1 It is the second leading cause of cancer-related deaths in women (15%) in the United States.1 Human epidermal growth factor receptor–2 (HER2/neu) is a protooncogene localized to chromosome 17. It encodes a transmembrane tyrosine kinase HER protein that aids in cell growth regulation and differentiation.2,3 The binding of ligand to the receptor complex on the cell surface leads to activation of intrinsic protein tyrosine kinase activity. This triggers a cascade of events leading to gene activation that results in mitogenic stimulation. The HER2/neu receptor, therefore, plays a key role in cellular growth.4 Overexpression of HER2/ protein or HER2/neu gene amplification is seen in various malignant neoplasms and correlates with specific clinical and histologic features. Overexpression occurs in 10% to 33% of breast cancer cases.5,6 This level of overexpression in node-positive patients correlates with poorer response to non-specific anticancer therapies, which suggests that targeted therapy with trastuzumab (Herceptin®) is appropriate for those patients.7-9

Recently, laboratory testing for HER2/neu has become more sensitive and specific. We examined the clinical impact of analytic uncertainty in testing for HER2/neu over a 5-year period in a community hospital.

Patients and methods

A retrospective analysis of our hospital’s tumor registry from 1998 to 2002 identified 248 patients with breast cancer and concomitant lymph node metastases. During this 5-year time period, testing for HER2/neu was not routine and was left to the physicians’ discretion when evaluating a patient for potential therapy with trastuzumab. The initial immunohistochemical (IHC) testing was performed with DakoCytomation’s rabbit antihuman c-erbB-2. For the study, IHC testing was repeated using the more recent NeoMarkers’ clone CB11, an antibody with increased specificity. The IHC testing methods used were in accordance with their respective manufacturer’s specification sheets.10,11 If a case previously tested positive with rabbit antihuman c-erbB-2 (a score of 3+) and now tested negative with clone CB11, showing discordance, our pathology department analyzed the results using fluorescence in situ hybridization (FISH).


The pathology reports showed that initial IHC testing with rabbit antihuman c-erbB-2 was performed on the primary lesion in 42 patients. Of these patients, 23 tested negative, and no further testing was undertaken. For the 19 patients who tested positive, HER2/neu analysis was repeated using clone CB11. Six of the 19 patients were found to be negative, for an initial discordance rate of 31%. Results for four of the six patients now testing negative with clone CB11 were confirmed by FISH analysis, indicating a true discordance rate of 21%.


Trastuzumab therapy prolongs survival in metastatic breast cancer patients whose tumors overexpress HER2/neu protein. Compared with chemotherapy alone, patients receiving a combination of chemotherapy and trastuzumab experience a significantly longer period of time until their disease progresses, have a higher rate of response to treatment, and respond to treatment for a longer duration. These effects are most marked in this first-line treatment of metastatic disease.12 Despite the theoretical benefits of such targeted treatment, not all patients respond favorably to trastuzumab. Among patients with HER2/neu-positive metastatic breast cancer resistant to conventional cytotoxic treatment, only about 25% benefit from adding trastuzumab to their cisplatin regimen.13,14 Trastuzumab is associated with some side effects, including cardiotoxicity. The overall incidence of cardiotoxicity is 4%, and for patients receiving concomitant therapy with an anthracycline, the incidence increases to 27%, of which 16% develop class III/IV heart failure based on the New York Heart Association classification.15,16 It has also been suggested that trastuzumab’s cardiotoxicity may be higher in patients who have a history of exposure to anthracycline.17

The 31% discordance rate observed in our small study group suggests that resolving analytic uncertainty may be important, considering the toxicity risk and costs associated with targeted therapy. Newer methods of HER2/neu testing open a window of uncertainty for patients who are prescribed treatment based on the results of older methods of testing. Our study suggests that patients deemed eligible for targeted therapy using earlier HER2/neu testing methods may not be eligible when current techniques are applied. Despite the inherent limitations related to the small size of our study, there do appear to be treatment-related issues based on previous testing methods. When IHC testing is used alone there is a discrepancy in IHC-positive staining (a score of 3+) and a patient’s response to trastuzumab. When FISH assay was added as a means for confirmation, a clearly improved response rate (54% versus 30%) was seen in patients whose positivity was confirmed using this test.18 In addition, the interpretation of IHC staining is subjective and may be affected by the experience of the interpreting pathologist. Current data suggest that with existing IHC methods and a well-trained pathologist, results are 80% to 85% reliable.19 When considering this relatively low level of reliability in conjunction with the costs and toxicity of trastuzumab, it is apparent that the most sensitive and specific methods available should be used.


Our institution is fortunate to have pathologists trained in conducting FISH analysis for confirmatory testing of HER2/neu status. Other community hospitals may lack the technical ability to perform the more accurate FISH analysis. The possibility of inaccurate assessment of a patient’s HER2/neu status is a real concern, and it may be necessary for some hospitals to refer archived material to outside institutions for confirmatory analysis.


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10. Polyclonal Rabbit Anti-human c-erbB-2 Oncoprotein [package insert]. Carpinteria, Calif: DakoCytomation, Inc; 2005.

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14. Simon R, Nocito A, Hubscher T, et al. Patterns of HER2/neu amplification and overexpression in primary and metastatic breast cancer. J Natl Cancer Inst. 2001;93(15): 1141-1146.

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16. Herceptin® [package insert]. Switzerland: F. Hoffmann-LaRoche Limited; 2000.

17. Robert NJ. Trastuzumab-associated cardiotoxicity. Breast Cancer Update for Surgeons [serial on CD-ROM]. 2004;3(3): CD-2/Track 5.

18. Mass RD, Press M, Anderson S, et al. Improved survival benefit from Herceptin® in patients selected by fluorescent in situ hybridization (FISH). Paper presented at: Annual Meeting of the American Society of Clinical Oncology; May 12-15, 2001; San Francisco, Calif.

19. Alfred DC. Practical approach to HER2 testing in community practice. Breast Cancer Update for Surgeons [serial on CD-ROM]. 2004;3(4):CD-2/Track 26.