Purpose: The present pilot study was performed to elucidate whether early changes in serum Her-2/neu extracellular domain (ECD) levels during trastuzumab-based treatment would predict the clinical course of disease in patients with metastatic breast cancer.

Experimental Design: Sera from 55 patients with Her-2/neu-overexpressing metastatic breast cancer obtained immediately before each weekly administration of trastuzumab were analyzed by a serum Her-2/neu ELISA.

Results: Whereas response rates were significantly higher in patients with elevated (≥15 ng/ml) ECD levels before initiation of treatment (35% versus 7%, P = 0.045), progression-free and overall survival did not differ significantly between patients with normal and elevated ECD levels. In patients responding to treatment, ECD levels decreased significantly as early as from day 8 of treatment onwards (all P for weekly measurements versus baseline <0.001). In contrast, no significant change in ECD levels was observed in patients with progressive disease. Multiple logistic regression analyses identified kinetics of ECD levels as the only factor that allowed for the accurate prediction of response likelihood as early as from day 8 of trastuzumab-based treatment onwards (P = 0.020). In addition, determination of serial ECD levels allowed for the prediction of the risk for disease progression within the observed period as early as day 15 of treatment (P = 0.010).

Conclusions: Serial monitoring of the ECD may represent a valuable tool for early prediction of the probability of response and progression-free survival to trastuzumab-based treatment and is thus likely to contribute to an optimization of treatment and resource allocation.

The human epidermal growth factor (Her-2/neu) oncoprotein is composed of an intracellular tyrosine kinase portion, a short transmembrane section, and an extracellular ligand-binding domain (ECD, p105). The latter is frequently cleaved from the cellular surface of Her-2/neu-overexpressing cells resulting in ligand-independent receptor activation and downstream signaling (1, 2, 3, 4, 5). Overexpression of the Her-2/neu protein arises predominantly through amplification of the c-erbB-2/neu oncogene and occurs in 25–30% of breast carcinomas. Both Her-2/neu overexpression and increased serum levels of the Her-2/neu ECD, which can be observed in up to 70% of women with Her-2/neu-overexpressing metastatic breast cancer (depending on the intensity of membranous Her-2/neu overexpression, tumor mass, and metastatic pattern; Refs. 6, 7) have frequently been associated with increased biological aggressiveness including poorer response rates to alkylating agent-based chemotherapy and endocrine therapy resulting in shorter progression-free and overall survival in this patient cohort (6, 7, 8, 9).

Trastuzumab, a monoclonal antibody targeting the Her-2/neu oncoprotein has not only demonstrated objective responses when administered as a single agent (6, 10, 11, 12), but its combined use with chemotherapy has also prolonged progression-free and overall survival as compared with standard treatment in patients with Her-2/neu-overexpressing metastatic breast cancer (9, 13, 14, 15, 16). However, the introduction of this treatment modality has caused concern about the availability of appropriate economic resources. With approximately 170,000 cases of breast cancer being diagnosed in the United States and in the European Union per year and major adjuvant treatment trials with trastuzumab underway, the use of trastuzumab is increasingly important for both patients and health economists. To date, further predictive factors for response to trastuzumab-based therapy apart from tumor characteristics of Her-2/neu protein overexpression or oncogene amplification, which would allow for the more targeted use of this treatment modality either as single agent or in combination with cytotoxic chemotherapy, are lacking. Therefore, we have attempted to identify such a factor by investigating the predictive potential of the serum Her-2/neu ECD. This assumption was based on previous experiments from our laboratory that have demonstrated that the Her-2/neu ECD competes with full-length p185 in binding to anti-Her-2/neu antibodies, thus neutralizing the biological effects of trastuzumab in vitro(17, 18). Results from our laboratory also corroborate other clinical studies, which have demonstrated a significantly decreased plasma half-life of trastuzumab in patients with high serum levels of Her-2/neu ECD (6, 9), and demonstrate the potential for interference of the Her-2/neu ECD with the biological effects of trastuzumab in vivo.

By extending these trials, we have now performed the present pilot study using serial measurements of serum Her-2/neu ECD to elucidate the predictive value of ECD baseline values and of early changes of serum ECD levels during weekly trastuzumab-based therapy.

We report that early changes in serum ECD levels during treatment with trastuzumab predict both subsequent response and progression-free survival to trastuzumab-based treatment.

Eligibility.

Potentially eligible patients had Her-2/neu-overexpressing (immunohistochemistry 2+ or 3+ as determined by the HercepTest; DAKO Diagnostics, Austria) metastatic breast cancer and were scheduled to receive trastuzumab (Herceptin; Roche Pharmaceuticals, Vienna, Austria) ± chemotherapy in accordance with previously published treatment protocols (6, 9, 11, 13, 14, 16, 19, 20) at the discretion of the treating physician. All study participants had bi-dimensionally measurable (with both diameters >1.0 cm and at least one diameter >1.5 cm) disease (excluding previously irradiated lesions) with clearly defined margins and radiologically (computed tomography and/or magnetic resonance imaging and/or ultrasound) documented tumor progression during the preceding 3 weeks. An interval of at least 4 weeks and recovery from all preceding treatment-related toxicities (excluding alopecia) was required between completion of prior therapy and study entry. Data on estrogen and progesterone receptor status of tissue samples from which the original assessment of Her-2/neu overexpression had been performed were available from pathology records. Patients who had previously received treatment with monoclonal antibodies, tumor vaccines, or biological-response modifiers were not eligible. Further inclusion criteria were Eastern Cooperative Oncology Group performance status 0–2, age ≥19 years, estimated life expectancy ≥12 weeks. In accordance with our institutional ethical committee guidelines, signed informed consent to participate in the present study was obtained from all patients; before each week, 8 ml of blood were drawn from the same venous access subsequently used for infusion of trastuzumab. Further exclusion criteria were bone metastases as the only site of active disease, second malignancy with the exception of in situ cervical cancer, adequately treated basal cell or squamous cell carcinoma of the skin, history of congestive heart failure (unless medically controlled), myocardial infarction within the last 2 years, active infection, altered mental status that would prohibit the understanding and the giving of informed consent, pregnancy, or breast feeding. In case of concomitant treatment with chemotherapy, patients with inadequate hematological function as defined by WBCs < 2.0 × 109/liter, absolute neutrophil count < 1.5 × 109/liter, platelets < 50 × 109/liter, severe hepatic or renal dysfunction (bilirubin > 2.0× or aspartate aminotransferase > 3.0× upper limits of normal, creatinine clearance < 30 ml/min) were not eligible.

Treatments.

Trastuzumab (4 mg/kg of body weight i.v. loading dose for 90 min followed by a weekly 2 mg/kg maintenance dose for 30 min, as described previously; Ref. 6) was administered until evidence of disease progression, consent withdrawal, or toxicity prompting cessation of treatment. In addition, patients were allowed to receive chemotherapy according to published protocols (9, 13, 14, 19) immediately after trastuzumab at the discretion of their treating physician. After completion of six to eight treatment cycles, patients achieving an objective response or disease stabilization to trastuzumab plus chemotherapy were offered maintenance therapy with single-agent trastuzumab until disease progression. Cardiac safety was routinely monitored by echocardiography every other month during treatment and if clinically indicated. Treatment was withheld until resolution of symptoms in patients experiencing grade ≥ 2 hematological and/or grade ≥ 3 nonhematological toxicity (with the exception of alopecia) according to National Cancer Institute Common Toxicity Criteria. In addition, patients experiencing grade 4 hematological or grade ≥ 3 nonhematological (with the exception of alopecia) toxicity or a decrease in cardiac-resting ejection fraction of ≥20% of baseline value or to less the lower normal limit went off study. If absolute neutrophil count on the day of scheduled treatment was below 1.5 × 109/liter, chemotherapy was withheld until resolution of neutropenia. Patients with prolonged hematological recovery (>3 weeks) went off study.

Acquisition and Analysis of Serum Probes.

Blood was drawn into native tubes immediately before each infusion of trastuzumab and on the date of clinical evaluation of patient response to treatment, centrifuged, and the resulting sera was analyzed using a sequential solid phase sandwich human Her-2/neu quantitative ELISA (Her-2/neu Microtiter ELISA; Oncogene Science, Cambridge MA) according to the manufacturer’s instructions. Microplates were washed using an automated washer (Dias Microplate Washer; Dynex Technologies, Denkendorf, Germany). Absorbance reading was performed using an automated reader (FLUOstar Galaxy; BMG Labtech GmbH, Offenburg, Germany) and calculated using the Fluoscan Galaxy software (versions 4.20–0; BMG Labtech GmbH). Intra- and interassay precision of the assay were <5% coefficient of variation. No cross-reactivity of the assay with epidermal growth factor receptor, trastuzumab, human antimouse antibodies, endogenous substances, vitamins, over-the-counter drugs, or antineoplastic agents has been found (21).

Lesion Measurement and Evaluation of Response to Treatment.

Restaging was performed every 8 weeks (or earlier if disease progression was clinically evident). Response evaluation and confirmation was performed in accordance with the Southwest Oncology Group response criteria and end-point definitions (22). In brief, complete response was defined as a complete disappearance of any tumor-related symptoms and all lesions in imaging studies, without appearance of any new lesions lasting for at least 4 weeks. Partial response was defined as >50% decrease under baseline in the sum of products of perpendicular diameters of all measurable lesions and at least stabilization of all nonmeasurable lesions lasting for a minimum of 4 weeks. Progressive disease was defined as a >25% increase in the sum of products of all measurable lesions, an unequivocal increase of nonmeasurable disease, or the appearance of new lesions. Disease was classified as being stable if no criteria for classifying responses as complete response, partial response, or progressive disease were met. For subsequent analyses, patients experiencing disease stabilization lasting >6 months were grouped together with patients achieving an objective response to treatment into a “clinical benefit group.”

Statistical Analysis.

Frequencies of patients’ characteristics were compared by Fisher’s exact test. Wilcoxon-signed ranks test was used for comparisons between ECD measurements at baseline and throughout treatment within and between the groups of patients experiencing a response, clinical benefit, or disease progression to trastuzumab.

In patients with elevated serum Her-2/neu levels before initiation of treatment, multivariate analyses were used to determine whether changes in concentrations of serum ECD could predict response, clinical benefit, progression-free survival, and overall survival from trastuzumab-based treatment. Grade of Her-2/neu overexpression (2+ versus 3+), patient age, recurrence-free interval (duration from initial diagnosis to appearance of metastatic disease), anthracycline pretreatment (for early or metastatic breast cancer), number of prior chemotherapeutic regimens for metastatic disease, estrogen receptor and progesterone receptor status, performance status, sites of active disease (visceral versus soft tissue only), number of organs involved by metastatic disease, type of treatment (single-agent trastuzumab versus combination with chemotherapy) were entered as potential confounders. Multiple logistic regression analyses were used to determine whether any of these variates could predict response or clinical benefit; multiple Cox regression models were used to identify the properties of the predictors mentioned above on progression-free and overall survival. Confounders without significant influences were removed by the backward selection method based on the Wald statistic. The related risk, the odds ratio, and 95% confidence interval (CI) were calculated with the proportional hazard method with respect to logistic regression and Cox regression, respectively. Survival curves (progression-free and overall survival) were compared with the log-rank test. For all analyses, a P < 5% was considered statistically significant. SPSS statistical software system (SPSS Inc., Chicago, IL, version 10.0) was used for all calculations.

Study Population.

Between April 2000 and July 2001 a total of 55 patients (median age, 53.0; range 27.6 to 81.0 years) with Her-2/neu-overexpressing metastatic breast cancer were enrolled. An overview of patients’ characteristics and corresponding baseline ECD values is depicted in Tables 1 and 2. During the study period, a total of 885 infusions of trastuzumab were performed. Sera for Her-2/neu measurements were obtained before each infusion. All patients were evaluable for response. Overall, six complete and nine partial responses (objective-response rate 27%)—all of which occurred in patients with grade 3+ Her-2/neu-overexpressing tumors—were observed (P for response according to the grade of Her-2/neu overexpression, 0.052). Disease stabilization lasting >6 months was achieved in 13 (24%) patients (including 2 of 9 patients with grade 2+ Her-2/neu-overexpressing tumors), whereas 27 (49%) patients (including 7 of 9 patients with grade 2+ overexpressing tumors) progressed despite treatment (P for clinical benefit according to the grade of Her-2/neu overexpression, 0.078). Objective responses were observed in 1 of 8 (13%) patients receiving single-agent trastuzumab and 14 of 47 (30%) patients additionally receiving chemotherapy (P = 0.040); clinical benefit was achieved in 3 of 8 (38%) patients treated with trastuzumab only and 25 of 47 (53%) patients additionally receiving chemotherapy (P = 0.469).

The present analysis covers a median observation period of 26.4 (range 20.7 to 39.5) months. Treatment was discontinued in 49 patients because of progressive disease (n = 46), hematological (n = 1), or cardiac (n = 1) toxicity or consent withdrawal (n = 1). Median progression-free survival calculated from the survival function was 4.9 (range 0.7–32.6) months. As of June 2003, 30 (55%) deaths were observed, all of which were attributed to disease progression. Median overall survival calculated from the survival function was 19.9 (range 1.7–38.5) months.

Baseline Serum Her-2/neu ECD Levels.

The median (range) baseline serum ECD levels was 45.1 (range 5.2–6076.2) ng/ml. As defined by the upper normal limit of 15 ng/ml (7, 23, 24, 25), a total of 40 (73%) patients presented with increased serum levels suggesting tumoral shedding of the Her-2/neu ECD into the bloodstream. As shown in Table 2, patients with grade 3+ Her-2/neu tumoral overexpression had significantly higher baseline ECD values (median 53.4, range 5.2–6076.2 ng/ml) than those with grade 2+ overexpression (median 12.1, range 9.3–19.1 ng/ml, P = 0.002). Patients with liver and/or lung metastases (median 61.4, range 9.2–6076.2 ng/ml) had significantly higher ECD levels at baseline, as compared with patients without visceral involvement (median 11.9, range 5.2–49.7 ng/ml, P < 0.001).

Impact of Baseline ECD Levels on Response, Progression-Free, and Overall Survival.

Response rates in patients with elevated (≥15 ng/ml) baseline serum ECD levels were significantly higher (35%, 14 of 40) as compared with the response rate observed in patients with normal (<15 ng/ml) baseline ECD values before initiation of treatment (7%, 1 of 15; P = 0.045). Likewise, responding patients had significantly higher absolute baseline ECD values (median 107.0, range 11.9–230.0 ng/ml) as compared with patients with progressive disease (median 17.4, range 5.2–6076.2 ng/ml, P = 0.028). The rates of clinical benefit were similar between patients with elevated (22 of 40, 55%) and patients with normal ECD levels at baseline (6 of 15, 40%; P = 0.375). Likewise, absolute baseline ECD levels of patients experiencing a clinical benefit (median 53.2, range 6.1–245.4 ng/ml) did not differ significantly from those observed in patients with progressive disease (median 17.4, range 5.2–6076.2 ng/ml, P = 0.195).

Median (range) progression-free survival was 5.7 (0.7–28.4) months in patients with elevated baseline ECD values and 4.5 (0.9–32.6) months in those with normal ECD levels (P = 0.813). Median (range) overall survival in patients with elevated baseline ECD levels was 17.4 (2.1–32.9) months, which did not differ significantly from the overall survival observed in patients with baseline ECD values <15 ng/ml (median 27.0, range 7.2–38.5 months, P = 0.410). In multivariate Cox regression analyses, there was a trend toward longer progression-free (P = 0.099) and overall survival (P = 0.060) in patients with higher absolute baseline ECD values and toward longer overall survival in patients with grade 3+ overexpressing tumors (P = 0.066).

Kinetics of Serum Her-2/neu ECD Concentrations During Trastuzumab-Based Treatment.

Kinetics of serum ECD levels (expressed as % of baseline values) in the course of treatment are depicted grouped according to the observed responses in Fig. 1. A significant decrease of ECD levels was observed in patients experiencing an objective response or clinical benefit from trastuzumab-based therapy as early as from day 8 of treatment onwards (all Ps for weekly measurements versus baseline were <0.001 in both the total collective and in patients with baseline values ≥15 ng/ml, respectively). In contrast, no significant change in ECD levels was observed in patients with progressive disease (all Ps > 0.05).

For instance, on day 15 of treatment, patients progressing despite therapy had a mean (±SD) decrease in serum Her-2/neu ECD levels of 11.5 ± 31.4%. Patients experiencing a clinical benefit had a mean decrease of 41.5 ± 20.9%, and patients responding to treatment had a mean decrease of 49.5 ± 20.8%. The magnitude of all these changes was much larger than the coefficient of variation of the assay.

Prediction of Response Probability by Determination of Serum Kinetics of the ECD.

Patients with elevated baseline ECD levels (n = 40) were entered into multiple logistic regression analyses to calculate the odds ratio of response and clinical benefit predicted by early changes of ECD levels during trastuzumab-based treatment. In these analyses, changes in ECD concentrations in relation to baseline were the only predicting variables for the occurrence of response or clinical benefit with higher relative ECD concentrations predicting lower probabilities. Kinetics of serum ECD levels allowed for an accurate prediction of response probability to trastuzumab-based therapy as early as by day 8 after treatment (and onwards; all Ps < 0.05). The odds ratio and 95% CI for response corresponding to a 1% decrease in ECD levels observed on day 8 of treatment was 1.043 (1.007–1.080, P = 0.020); i.e., each 1% decrease of ECD values (from baseline values) observed on day 8 increased the probability of response by 4.3% (0.7–8.0%). The odds ratio (95% CI) for response corresponding to each 10% change of ECD levels in the course of treatment is shown in Table 3.

The prediction of probability of clinical benefit based on changes in ECD serum levels from baseline was possible from day 8 of treatment onwards (all Ps < 0.05; Table 3). Measuring early ECD changes the probability of response or clinical benefit could be predicted not only in the total cohort but also in patients with grade 3+ Her-2/neu-overexpressing tumors or patients receiving trastuzumab plus chemotherapy, the latter representing the largest patient subgroups (Table 3).

In addition, cut-point analyses for prediction of response and clinical benefit based on ECD levels (in % of baseline levels) observed on day 15 were performed (see receiver-operating characteristics curves in Fig. 2). For prediction of response, cut-point analysis revealed a cutoff at 66.1% of the baseline ECD concentration. Sensitivity and specificity were 0.833 and 0.722, respectively. The area below the curve was 0.856 (95% CI, 0.725–0.988; P = 0.001). Objective responses were observed in 10 of 15 (67%) patients whose ECD levels had dropped below 66.1% of baseline levels, whereas only 2 of 15 (13%) patients with ECD levels above 66.1% of baseline levels on day 15 responded.

For prediction of clinical benefit, cut-point analysis calculated a cutoff at 71.3% of the baseline ECD concentration (sensitivity 0.800, specificity 0.611). The area below the curve was 0.778 (95% CI, 0.630–0.925; P = 0.003). Thus, 16 of 23 (70%) patients with ECD levels below 71.3% of baseline levels on day 15 experienced a clinical benefit from trastuzumab-based treatment. In contrast, only 4 of 15 (27%) patients with ECD levels above 71.3% of pretreatment serum levels on day 15 experienced a clinical benefit.

Prediction of Progression-Free and Overall Survival by Serum Kinetics of the Her-2/neu ECD.

Multiple Cox regression analyses in patients with elevated baseline ECD levels revealed that early changes in ECD serum levels after initiation of trastuzumab-based treatment allowed for the prediction of progression-free survival. In these analyses, determination of the risk for disease progression within the observation period (median 26.4, range 20.7–39.5 months) was possible as early as from day 15 of trastuzumab-based treatment onwards; the related risk for progression within the observed period corresponding to a 1% increase of ECD values observed on day 15 of treatment was 1.018 (95% CI, 1.004–1.032; P = 0.010); i.e., each 1% increase of ECD values (from baseline values) observed on day 15 increased the risk for disease progression by 1.8% (95% CI, 0.4–3.2%). The probability values for progression-free survival corresponding to early changes in ECD values could not only be calculated within the total cohort but also in patients with grade 3+ Her-2/neu-overexpressing tumors and in patients receiving trastuzumab plus chemotherapy. None of the other patient characteristics analyzed was found to influence progression-free survival significantly. The related risk and 95% CI for disease progression corresponding to each 10% change in ECD levels from baseline values observed in the course of treatment are shown in Table 4. Survival curves comparing progression-free survival in patients whose ECD levels had increased on day 15 of treatment to those whose ECD levels had decreased are depicted in Fig. 3.

No significant prediction of the probabilities of overall survival could be made by any of the factors analyzed, probably because the majority of patients (43 of 55, 75%) had received further cytotoxic therapy after progression on trastuzumab-based treatment.

The weekly use of trastuzumab as either a single agent or in combination with cytotoxic chemotherapy has intensified the need for patients to undergo frequent therapeutic interventions as well as increased economic burden. Therefore, it seems a desirable goal to develop predictive indicators for response to treatment aside from tumoral Her-2/neu overexpression and chemotherapeutic pretreatment.

In the present study, we are able to confirm previous reports demonstrating that serum Her-2/neu ECD does not interfere with the biological effects of trastuzumab-based treatment in the majority of patients (6, 9). Our results indicate that patients with elevated serum ECD levels are more likely to respond to trastuzumab-based treatment. Furthermore, we report that in patients with elevated baseline ECD levels, changes of ECD levels throughout trastuzumab-based treatment do not only parallel (6, 9, 20) but even precede the clinical course of disease, thus allowing for a significant prediction of response, clinical benefit, and progression-free survival as early as after 1 and 2 weeks of treatment, respectively.

Within this context, it is interesting to note that serum ECD levels have not only been reported to correlate with the intensity of membranous Her-2/neu overexpression (6) and tumor burden (7) but that proteolytic cleavage of the ECD represents a ligand-independent activation mechanism leading to Her-2/neu phosphorylation and active Her-2/neu signaling (5). In our study, the higher-response rates observed in patients with elevated ECD levels may thus represent the increased sensitivity of tumors with more intense Her-2/neu expression and signaling toward trastuzumab-based treatment. Likewise, the rapid and profound decrease of ECD levels observed in patients responding to treatment may also represent inhibition of ECD release and inhibition of Her-2/neu activation in trastuzumab-sensitive tumors (5), then solely parallel the declining tumor mass.

We conclude, therefore, that serial measurements of the Her-2/neu ECD could have the potential to predict the clinical outcome of patients with Her-2/neu-overexpressing metastatic breast cancer treated with trastuzumab-based therapy. Thus, monitoring of serum Her-2/neu ECD levels should be incorporated in future, larger trials. The use of this predictive potential could not only focus and tailor treatment individually but also adapt to economic needs.

Grant support: Grant-in-aid from Oncogene Science/Bayer Diagnostics (USA/Germany) and Roche Pharmaceuticals (Austria).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Notes: R. N. is an employee of Bayer Immunodiagnostics, Leverkusen, Germany.

Requests for reprints: Wolfgang J. Köstler, Clinical Division of Oncology, Department of Medicine I, University Hospital of Vienna, 18-20 Waehringer Guertel, A-1090 Vienna, Austria. Phone: 43-1-40400-4445; Fax: 43-1-40400-4451; E-mail: wolfgang.koestler@akh-wien.ac.at

Fig. 1.

Dynamics of serum Her-2/neu extracellular domain (ECD) during trastuzumab-based treatment. Serial serum Her-2/neu levels in patients with elevated (≥15 ng/ml) baseline serum Her-2/neu ECD concentrations are depicted in accordance to the clinical course of disease (response, complete or partial response; benefit, response, or stable disease >6 months; PD, progressive disease). Measurements are expressed as % of the baseline value.

Fig. 1.

Dynamics of serum Her-2/neu extracellular domain (ECD) during trastuzumab-based treatment. Serial serum Her-2/neu levels in patients with elevated (≥15 ng/ml) baseline serum Her-2/neu ECD concentrations are depicted in accordance to the clinical course of disease (response, complete or partial response; benefit, response, or stable disease >6 months; PD, progressive disease). Measurements are expressed as % of the baseline value.

Close modal
Fig. 2.

Receiver-operating characteristics curves for response and clinical benefit in dependence on the relative Her-2/neu extracellular domain (ECD) concentration observed on day 15 of treatment in patients with elevated ECD levels at baseline. Cut-point analyses revealed a cutoff at 66.1 and 71.3% of the baseline ECD concentration for prediction of response and clinical benefit, respectively.

Fig. 2.

Receiver-operating characteristics curves for response and clinical benefit in dependence on the relative Her-2/neu extracellular domain (ECD) concentration observed on day 15 of treatment in patients with elevated ECD levels at baseline. Cut-point analyses revealed a cutoff at 66.1 and 71.3% of the baseline ECD concentration for prediction of response and clinical benefit, respectively.

Close modal
Fig. 3.

Kaplan-Meier functions of progression free survival in patients with elevated pretreatment serum Her-2/neu extracellular domain (ECD) levels comparing patients whose ECD levels had decreased (—, n = 32) with patients whose ECD levels had increased (- - -, n = 6) from baseline on day 15 of trastuzumab-based treatment (▪, censored cases, log-rank P < 0.001).

Fig. 3.

Kaplan-Meier functions of progression free survival in patients with elevated pretreatment serum Her-2/neu extracellular domain (ECD) levels comparing patients whose ECD levels had decreased (—, n = 32) with patients whose ECD levels had increased (- - -, n = 6) from baseline on day 15 of trastuzumab-based treatment (▪, censored cases, log-rank P < 0.001).

Close modal
Table 1

Patient characteristics

Patient characteristicn
Gender  
 Female 54 
 Male 
Chemotherapeutic pretreatment for metastatic disease  
 None 29 
 1 Regimen 15 
 ≥2 11 
Grade of Her-2/neu overexpression  
 Grade 2+ 
 Grade 3+ 46 
Treatment  
 Single-agent trastuzumab 
 Trastuzumab & paclitaxel 10 
 Trastuzumab & docetaxel 
 Trastuzumab & vinorelbine 28 
Sites of active disease  
 Liver 32 
 Bone 26 
 Lymph nodes 25 
 Lung 23 
 Skin 
 Other 18 
  
 Visceral metastases 43 
 Soft tissue disease only 12 
Patient characteristicn
Gender  
 Female 54 
 Male 
Chemotherapeutic pretreatment for metastatic disease  
 None 29 
 1 Regimen 15 
 ≥2 11 
Grade of Her-2/neu overexpression  
 Grade 2+ 
 Grade 3+ 46 
Treatment  
 Single-agent trastuzumab 
 Trastuzumab & paclitaxel 10 
 Trastuzumab & docetaxel 
 Trastuzumab & vinorelbine 28 
Sites of active disease  
 Liver 32 
 Bone 26 
 Lymph nodes 25 
 Lung 23 
 Skin 
 Other 18 
  
 Visceral metastases 43 
 Soft tissue disease only 12 
Table 2

Correlation of clinical characteristics with baseline serum Her-2/neu levels

Median (range) baseline ECDa (ng/ml)P
Grade 2+ Her-2/neu overexpression 12.1 (9.3–19.1)  
Grade 3+ Her-2/neu overexpression 53.4 (5.2–6076.2) 0.002 
Visceral involvement 61.4 (9.2–6076.2)  
Soft tissue disease only 11.9 (5.2–49.7) <0.001 
Median (range) baseline ECDa (ng/ml)P
Grade 2+ Her-2/neu overexpression 12.1 (9.3–19.1)  
Grade 3+ Her-2/neu overexpression 53.4 (5.2–6076.2) 0.002 
Visceral involvement 61.4 (9.2–6076.2)  
Soft tissue disease only 11.9 (5.2–49.7) <0.001 
a

ECD, extracellular domain.

Table 3

The odds ratios (OR) and 95% confidence intervals (95% CI) of response and clinical benefit (response or disease stabilisation >6 months) in dependence on early changes of Her-2/neu extracellular domain (ECD) during treatment with trastuzumab

The OR of response and clinical benefit for each 10% decrease of Her-2/neu ECD from baseline concentrations in the course of treatment are depicted for the total collective, patients with grade 3+ Her-2/neu-overexpressing tumors and patients receiving trastuzumab plus chemotherapy. Lower relative Her-2/neu ECD concentrations predict higher odds of response and benefit: i.e., in the total collective the OR of response doubles (1.989) in patients with an observed 10% decrease (from baseline) of ECD values on day 15, whereas the OR of response in patients with a 50% decrease on day 15 is 31.130 (1.9895). In analogy, the OR of response for a 50% increase of ECD values observed on day 15 is 0.032 [(1/1.989)5].

OR (95% CI) for response for each 10% decrease of ECD from baselinePOR (95% CI) for clinical benefit for each 10% decrease of ECD from baselineP
All patients     
 Day 8 1.519 (1.068–2.159) 0.020 1.365 (1.021–1.825) 0.036 
 Day 15 1.989 (1.139–3.471) 0.016 1.632 (1.131–2.357) 0.009 
 Day 22 2.052 (1.192–3.533) 0.010 1.649 (1.142–2.381) 0.008 
Patients with grade 3+ Her-2/neu overexpressing tumors     
 Day 8 1.475 (1.044–2.085) 0.027 1.359 (1.013–1.824) 0.041 
 Day 15 1.906 (1.114–3.261) 0.019 1.684 (1.133–2.502) 0.010 
 Day 22 2.199 (1.193–4.052) 0.012 1.721 (1.144–2.589) 0.009 
Patients receiving trastuzumab and concomitant chemotherapy     
 Day 8 1.622 (1.074–2.450) 0.021 1.531 (1.058–2.215) 0.024 
 Day 15 2.041 (1.123–3.711) 0.019 1.662 (1.094–2.525) 0.017 
 Day 22 2.0277 (1.130–3.640) 0.018 1.602 (1.085–2.365) 0.018 
OR (95% CI) for response for each 10% decrease of ECD from baselinePOR (95% CI) for clinical benefit for each 10% decrease of ECD from baselineP
All patients     
 Day 8 1.519 (1.068–2.159) 0.020 1.365 (1.021–1.825) 0.036 
 Day 15 1.989 (1.139–3.471) 0.016 1.632 (1.131–2.357) 0.009 
 Day 22 2.052 (1.192–3.533) 0.010 1.649 (1.142–2.381) 0.008 
Patients with grade 3+ Her-2/neu overexpressing tumors     
 Day 8 1.475 (1.044–2.085) 0.027 1.359 (1.013–1.824) 0.041 
 Day 15 1.906 (1.114–3.261) 0.019 1.684 (1.133–2.502) 0.010 
 Day 22 2.199 (1.193–4.052) 0.012 1.721 (1.144–2.589) 0.009 
Patients receiving trastuzumab and concomitant chemotherapy     
 Day 8 1.622 (1.074–2.450) 0.021 1.531 (1.058–2.215) 0.024 
 Day 15 2.041 (1.123–3.711) 0.019 1.662 (1.094–2.525) 0.017 
 Day 22 2.0277 (1.130–3.640) 0.018 1.602 (1.085–2.365) 0.018 
Table 4

The related risk (RR) and 95% confidence interval (95% CI) for disease progression depending on early changes of the serum Her-2/neu extracellular domain (ECD)

The RR is the factor by which the risk of disease progression within the observed period (median, 26.4; range 20.7–39.5 months) is multiplied, if ECD values (in % baseline) of treatment rise by 10% (depicted for the total collective, patients with grade 3+ Her-2/neu-overexpressing tumors and patients receiving trastuzumab plus chemotherapy). Lower relative serum Her-2/neu ECD values raise the probability of progression-free survival. For instance, in the total collective the RR for progression is 1.231 times higher in patients with an observed 10% increase (from baseline) of ECD values on day 22 and 2.827 times higher (1.2315) in patients with a 50% increase of ECD values over baseline observed on day 22, respectively. In contrast, the RR for progression with a 50% decrease of ECD values on day 22 is 0.354 (1/1.2315).

RR (95% CI) for progression for a 10% increase of ECD values
RR (95% CI)P
All patients   
 Day 8 1.033 (0.879–1.214) 0.691 
 Day 15 1.194 (1.043–1.367) 0.010 
 Day 22 1.231 (1.057–1.434) 0.008 
Patients with grade 3+ Her-2/neu overexpressing tumors   
 Day 8 1.037 (0.882–1.219) 0.662 
 Day 15 1.219 (1.057–1.405) 0.006 
 Day 22 1.240 (1.056–1.457) 0.009 
Patients receiving trastuzumab and concomitant chemotherapy   
 Day 8 1.038 (0.870–1.239) 0.678 
 Day 15 1.193 (1.013–1.405) 0.034 
 Day 22 1.230 (1.044–1.450) 0.013 
RR (95% CI) for progression for a 10% increase of ECD values
RR (95% CI)P
All patients   
 Day 8 1.033 (0.879–1.214) 0.691 
 Day 15 1.194 (1.043–1.367) 0.010 
 Day 22 1.231 (1.057–1.434) 0.008 
Patients with grade 3+ Her-2/neu overexpressing tumors   
 Day 8 1.037 (0.882–1.219) 0.662 
 Day 15 1.219 (1.057–1.405) 0.006 
 Day 22 1.240 (1.056–1.457) 0.009 
Patients receiving trastuzumab and concomitant chemotherapy   
 Day 8 1.038 (0.870–1.239) 0.678 
 Day 15 1.193 (1.013–1.405) 0.034 
 Day 22 1.230 (1.044–1.450) 0.013 

We thank Waclawa Kalinowski and Erika Marton for expert technical assistance and the Nurses of the Clinical Division of Oncology, Department of Internal Medicine I, University Hospital of Vienna for assistance. In addition, we thank Dr. Johann Blasina and Dr. Angelika Eiper for logistic support and Dr. Walter Carney for fruitful discussions.

1
Coussens L., Yang-Feng T. L., Liao Y. C., Chen E., Gray A., McGrath J., Seeburg P. H., Libermann T. A., Schlessinger J., Francke U., et al Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene.
Science (Wash. DC)
,
230
:
1132
-1139,  
1985
.
2
Brandt-Rauf P. W., Pincus M. R., Carney W. P. The c-erbB-2 protein in oncogenesis: molecular structure to molecular epidemiology.
Crit. Rev. Oncog.
,
5
:
313
-329,  
1994
.
3
Breuer B., DeVivo I., Luo J. C., Smith S., Pincus M. R., Tatum A. H., Daucher J., Minick C. R., Miller D. G., Nowak E. J., et al erbB-2 and myc oncoproteins in sera and tumors of breast cancer patients.
Cancer Epidemiol. Biomark. Prev.
,
3
:
63
-66,  
1994
.
4
Zabrecky J. R., Lam T., McKenzie S. J., Carney W. The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.
J. Biol. Chem.
,
266
:
1716
-1720,  
1991
.
5
Molina M. A., Codony-Servat J., Albanell J., Rojo F., Arribas J., Baselga J. Trastuzumab (herceptin), a humanized anti-Her2 receptor monoclonal antibody, inhibits basal and activated Her2 ectodomain cleavage in breast cancer cells.
Cancer Res.
,
61
:
4744
-4749,  
2001
.
6
Cobleigh M. A., Vogel C. L., Tripathy D., Robert N. J., Scholl S., Fehrenbacher L., Wolter J. M., Paton V., Shak S., Lieberman G., Slamon D. J. 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.
,
17
:
2639
-2648,  
1999
.
7
Lipton A., Ali S. M., Leitzel K., Demers L., Chinchilli V., Engle L., Harvey H. A., Brady C., Nalin C. M., Dugan M., Carney W., Allard J. Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer.
J. Clin. Oncol.
,
20
:
1467
-1472,  
2002
.
8
Yamauchi H., Stearns V., Hayes D. F. When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer.
J. Clin. Oncol.
,
19
:
2334
-2356,  
2001
.
9
Pegram M. D., Lipton A., Hayes D. F., Weber B. L., Baselga J. M., Tripathy D., Baly D., Baughman S. A., Twaddell T., Glaspy J. A., Slamon D. J. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment.
J. Clin. Oncol.
,
16
:
2659
-2671,  
1998
.
10
Pegram M., Slamon D. Biological rationale for HER2/neu (c-erbB2) as a target for monoclonal antibody therapy.
Semin. Oncol.
,
27
:
13
-19,  
2000
.
11
Vogel C. L., Cobleigh M. A., Tripathy D., Gutheil J. C., Harris L. N., Fehrenbacher L., Slamon D. J., Murphy M., Novotny W. F., Burchmore M., Shak S., Stewart S. J., Press M. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer.
J. Clin. Oncol.
,
20
:
719
-726,  
2002
.
12
Baselga J., Tripathy D., Mendelsohn J., Baughman S., Benz C. C., Dantis L., Sklarin N. T., Seidman A. D., Hudis C. A., Moore J., Rosen P. P., Twaddell T., Henderson I. C., Norton L. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer.
J. Clin. Oncol.
,
14
:
737
-744,  
1996
.
13
Slamon D. J., Leyland-Jones B., Shak S., Fuchs H., Paton V., Bajamonde A., Fleming T., Eiermann W., Wolter J., Pegram M., Baselga J., Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.
N. Engl. J. Med.
,
344
:
783
-792,  
2001
.
14
Burstein H. J., Kuter I., Campos S. M., Gelman R. S., Tribou L., Parker L. M., Manola J., Younger J., Matulonis U., Bunnell C. A., Partridge A. H., Richardson P. G., Clarke K., Shulman L. N., Winer E. P. Clinical activity of trastuzumab and vinorelbine in women with HER2-overexpressing metastatic breast cancer.
J. Clin. Oncol.
,
19
:
2722
-2730,  
2001
.
15
Burris H. A., III Docetaxel (Taxotere) plus trastuzumab (Herceptin) in breast cancer.
Semin. Oncol.
,
28
:
38
-44,  
2001
.
16
Miller K. D., Sisk J., Ansari R., Gize G., Nattam S., Pennington K., Monaco F., Sledge G. W., Jr. Gemcitabine, paclitaxel, and trastuzumab in metastatic breast cancer.
Oncology (Huntingt.)
,
15
:
38
-40,  
2001
.
17
Brodowicz T., Wiltschke C., Budinsky A. C., Krainer M., Steger G. G., Zielinski C. C. Soluble HER-2/neu neutralizes biologic effects of anti-HER-2/neu antibody on breast cancer cells in vitro.
Int. J. Cancer
,
73
:
875
-879,  
1997
.
18
Krainer M., Brodowicz T., Zeillinger R., Wiltschke C., Scholten C., Seifert M., Kubista E., Zielinski C. C. Tissue expression and serum levels of HER-2/neu in patients with breast cancer.
Oncology
,
54
:
475
-481,  
1997
.
19
Seidman A. D., Fornier M. N., Esteva F. J., Tan L., Kaptain S., Bach A., Panageas K. S., Arroyo C., Valero V., Currie V., Gilewski T., Theodoulou M., Moynahan M. E., Moasser M., Sklarin N., Dickler M., D’Andrea G., Cristofanilli M., Rivera E., Hortobagyi G. N., Norton L., Hudis C. A. Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification.
J. Clin. Oncol.
,
19
:
2587
-2595,  
2001
.
20
Esteva F. J., Valero V., Booser D., Guerra L. T., Murray J. L., Pusztai L., Cristofanilli M., Arun B., Esmaeli B., Fritsche H. A., Sneige N., Smith T. L., Hortobagyi G. N. Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer.
J. Clin. Oncol.
,
20
:
1800
-1808,  
2002
.
21
Payne R. C., Allard J. W., Anderson-Mauser L., Humphreys J. D., Tenney D. Y., Morris D. L. Automated assay for HER-2/neu in serum.
Clin. Chem.
,
46
:
175
-182,  
2000
.
22
Green S., Weiss G. R. Southwest Oncology Group standard response criteria, endpoint definitions and toxicity criteria.
Investig. New Drugs
,
10
:
239
-253,  
1992
.
23
Cook G. B., Neaman I. E., Goldblatt J. L., Cambetas D. R., Hussain M., Luftner D., Yeung K. K., Chan D. W., Schwartz M. K., Allard W. J. Clinical utility of serum HER-2/neu testing on the Bayer Immuno 1 automated system in breast cancer.
Anticancer Res.
,
21
:
1465
-1470,  
2001
.
24
Molina R., Jo J., Filella X., Zanon G., Pahisa J., Munoz M., Farrus B., Latre M. L., Gimenez N., Hage M., Estape J., Ballesta A. M. C-erbB-2 oncoprotein in the sera and tissue of patients with breast cancer. Utility in prognosis.
Anticancer Res.
,
16
:
2295
-2300,  
1996
.
25
Molina R., Jo J., Filella X., Zanon G., Pahisa J., Munoz M., Farrus B., Latre M. L., Escriche C., Estape J., Ballesta A. M. c-erbB-2 oncoprotein, CEA, and CA 15.3 in patients with breast cancer: prognostic value.
Breast Cancer Res. Treat.
,
51
:
109
-119,  
1998
.