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Technical Briefs |
1 Department of Gynecology and Obstetrics, University of Cologne, 50924 Cologne, Germany;
2
Bayer Vital, 51368 Leverkusen, Germany;
aaddress correspondence to this author at: Department of Gynecology and Obstetrics, University of Cologne, Kerpener Strasse 34, 50924 Cologne, Germany; fax 49-221-478-7495, e-mail thomas.schoendorf{at}medizin.uni-koeln.de
The HER-2/neu oncoprotein, a member of the epidermal growth factor receptor family, is overexpressed in 20–30% of breast cancer patients as a result of gene amplification (1)(2). Various methods have been used to quantify both HER-2/neu gene amplification and its gene product. Those methods most clinically applicable include fluorescence in situ hybridization for gene amplification and immunohistochemistry or ELISAs for protein overexpression (3)(4). The amplification/overexpression of HER-2/neu may have several clinical implications that indicate an aggressive form of breast cancer with a significantly shortened relapse-free interval (1)(2). Nevertheless, the independence of this factor remains uncertain, as does the predictive value of HER-2/neu overexpression for therapeutic success (5).
Treatment with trastuzumab, a humanized monoclonal antibody that binds with high affinity to the extracellular domain of HER-2/neu, is a promising therapy for metastatic breast cancers that overexpress HER-2/neu protein. In experimental models of breast cancer, trastuzumab was active in vitro and in vivo (5)(6); in addition, the administration of trastuzumab in combination with chemotherapy showed an augmented antitumor effect (7)(8). The largest clinical study recruited 222 patients with HER-2/neu-overexpressing metastatic breast cancer who had relapsed after one or two chemotherapy regimens (9). The overall response rate (complete response plus partial response), as determined by an independent Response Evaluation Committee, was 16%, with a 4% complete response and a 12% partial response rate. In 469 patients with HER-2/neu-overexpressing metastatic breast cancer who had not been previously treated with chemotherapy, the combination of trastuzumab with chemotherapy was associated with a longer time to disease progression, a higher rate of objective response, a longer duration of response, a lower death rate after 1 year, longer survival, and a 20% reduction in the risk of death (2). A metaanalysis comprising 22 616 patients in 97 studies confirmed the shortened relapse-free interval and decreased survival rate of breast cancer patients with HER-2/neu-overexpressing tumors (10).
No data are available on an objective laboratory marker to monitor the therapeutic success of trastuzumab therapy alone or in combination with chemotherapy. We studied a serum assay for HER-2/neu for its ability to predict response to trastuzumab treatment during longitudinal follow-up.
The extracellular domain of the HER-2/neu protein is proteolytically shed from HER-2/neu-overexpressing tumor tissues into the blood (4), increasing plasma HER-2/neu (11)(12).
We studied 23 trastuzumab patients whose tumors highly expressed the HER-2/neu antigen (immunohistochemical score, 3+): 19 patients had metastases, 1 had a local relapse; and 3 were in an adjuvant treatment situation. All patients were treated by mastectomy and axillary lymph node dissection. Histologically, all tissues displayed the invasive ductal subtype. The clinical data for the patients group are summarized in Table 1
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All plasma samples were obtained subsequent to standard hematologic procedures. Serologic HER-2/neu concentrations were determined using the HER-2/neu method from Bayer Diagnostics (13). Data were obtained automatically by the Bayer Immuno 1TM immunoanalyzer. In brief, a fluorescein-labeled (NB-3) antibody and a alkaline phosphatase-labeled (TA-1) monoclonal antibody were used to bind the extracellular domain of HER-2/neu. The complex was separated by magnetic particles coated with anti-fluorescein antibodies. p-Nitrophenyl phosphate was used as substrate. Each assay included HER-2/neu calibrators ranging from 0 to 250 µg/L. Samples with results above this range were diluted and measured again. Each assay was performed in duplicate.
Parallel to the HER-2/neu determination we measured CA 15-3 in the plasma samples on the Immuno 1 with reagents from Bayer Diagnostics.
For statistical analysis, we used SPSS 9.0 for Windows (SPSS). The data for both tumor markers were analyzed for deviation from the norm. Although a cutoff of 15 µg/L for HER-2/neu determinations has been suggested by most researchers, a clear definition remains to be elucidated based on larger cohorts of patients in additional studies. In our study, a cutoff of 20 µg/L was more appropriate. The choice was influenced by the fact that we examined plasma rather than serum, which was used in the other studies. In addition, only patients with concentrations exceeding 20 µg/L showed a relevant change in HER-2/neu.
For patients with increased HER-2/neu, the results were further analyzed to determine whether the change in tumor marker concentrations paralleled the clinical course. The clinical course was defined by imaging diagnostics in objective terms according to the Union Internationale Contre le Cancer criteria as remission or progression of the disease. For individualization of the analyses, a change in concentration was defined as an increase or decrease 
10% of the first value and a minimum rate exceeding 10 µg/L per 4 weeks.
All plasma samples could be successfully analyzed. Plasma HER-2/neu concentrations were 3.2–5130 µg/L. The SD of duplicates never exceeded 0.8% of each mean value.
The median observation time was 13 months with a range between 4 and 22 months (Table 1
). At a cutoff of 20 µg/L, HER-2/neu was increased in 12 of 19 patients (63%), whereas CA 15-3 was increased in 18 of 19 (95%). All but 1 of the 12 patients with increased HER-2/neu had visceral metastases. In the group that did not have increased HER-2/neu, 5 of 7 patients (71%) developed osseous metastases. Eleven of 13 patients (85%) with metastases localized in the liver had increased HER-2/neu.
In the group with increased HER-2/neu, 35 clinical events (remission or progression according to Union Internationale Contre le Cancer criteria) were documented. Changes of HER-2/neu correlated with the clinical event in 74% of the events (26 of 35). The CA 15-3-sensitive group showed a similar correlation (31 of 42 clinical events; 74%). In the four patients without evidence of metastases, both HER-2/neu and CA 15-3 were below their cutoff values.
Shown in Fig. 1
are three examples of the longitudinal HER-2/neu concentration in plasma samples in comparison with the CA 15-3 concentration and the clinical course of the patients. Fig. 1C
displays the course for a patient with visceral metastases and increased HER-2/neu who could be monitored only by HER-2/neu determinations. In the data supplement (available with the online version of this Technical Brief at http://www.clinchem.org/content/vol48/issue8/), Fig. 1A
illustrates results for a patient with a serologic HER-2/neu value <20 µg/L (patient 15), and Fig. 1B
illustrates the course for a patient with visceral metastases and an increased HER-2/neu concentration who could be monitored by both plasma HER-2/neu and CA 15-3 determinations (patient 13).
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Serum HER-2/neu may be useful for monitoring of response to trastuzumab (11)(12). The serologic HER-2/neu determination may identify patients with a worse prognosis better than approaches using immunohistochemistry or fluorescence in situ hybridization (14).
Increased serum HER-2/neu was detected in 12% of an overall breast cancer group (15) and provided independent prognostic information (12) that was superior to that from nodal involvement or hormone receptor status (15). Preoperative HER-2/neu concentrations in sera are significantly associated with the tissue marker (16). Thus, it seemed opportune to reduce further explorative studies on HER-2 serology to patients with HER-2/neu-overexpressing tissues. However, the main investigations were performed without selecting a subgroup of patients receiving trastuzumab therapy. We here present the first study of the longitudinal course of serologic HER-2/neu values in patients receiving trastuzumab.
The HER-2/neu values were detected using a HER-2/neu method on the Bayer Immuno 1 system. We confirmed that the data collection was fast and reproducible and could be incorporated into clinical routine easily (13)(17). Both serum and plasma are acceptable samples (18).
Retrospectively, HER-2/neu testing indicated remission or disease progression in 74% of all cases. The sensitivity increased when the patients were restricted to metastatic breast cancer patients with visceral metastases. However, increased serum concentrations have been reported in benign diseases, including liver cirrhosis and hepatitis (19). One question that remains unanswered is whether the high sensitivity in breast cancer with liver metastases reflects an acquired liver dysfunction or whether HER-2/neu-overexpressing breast tumor cells preferentially affect the liver rather than bones or the lung. Theoretically, the environment of bone and lung may induce a decrease in HER-2/neu expression. Taken together, HER-2/neu appears not to be a specific breast cancer marker (20), but because of its sensitivity, the serologic HER-2/neu determination may provide an additional tool to monitor trastuzumab therapy of breast cancer patients. Accordingly, serologic HER-2/neu determinations might be useful to improve outcome by avoiding time lost through ineffective regimens and by avoiding exacerbated side effects and unnecessary costs.
In summary, plasma HER-2/neu parallels the clinical course in patients with metastatic breast cancer, especially in patients with visceral metastases.
Acknowledgments
We thank Thérèse Tanasale for excellent technical assistance and Sibylle Fechner for preparing the graphs.
Footnotes
1 both authors contributed equally to this work; 
References
The following articles in journals at HighWire Press have cited this article:
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  W. P. Carney, R. Neumann, A. Lipton, K. Leitzel, S. Ali, and C. P. Price Potential Clinical Utility of Serum HER-2/neu Oncoprotein Concentrations in Patients with Breast Cancer Clin. Chem., October 1, 2003; 49(10): 1579 - 1598. [Abstract] [Full Text] [PDF]
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