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European Journal of Heart Failure 2007 9(2):173-177; doi:10.1016/j.ejheart.2006.05.010
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© 2007 European Society of Cardiology

Serum HER2 levels are increased in patients with chronic heart failure

Patrick J. Perika,b, Elisabeth G.E. de Vriesb, Jourik A. Gietemab, Winette T.A. van der Graafb, Tom D.J. Smildea, Dirk T. Sleijferb and Dirk J. van Veldhuisena,*

a Department of Cardiology, Thorax Center, University of Groningen and University Medical Center Groningen P.O. Box 30 001, 9700 RB Groningen, The Netherlands
b Department of Medical Oncology of the University of Groningen and University Medical Center Groningen The Netherlands

* Corresponding author. Tel.: +31 50 361 6161; fax: +31 50 361 4391. E-mail address: d.j.van.veldhuisen{at}thorax.umcg.nl


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: The use of trastuzumab, an antibody against the human epidermal growth factor receptor 2 (HER2), in patients with HER2 positive metastatic breast cancer, is related to cardiotoxicity.

Aims: To investigate whether serum HER2 is increased in heart failure patients and related to disease severity.

Methods: Serum HER2, plasma tumor necrosis factor (TNF)-{alpha} and its soluble (s) receptors (sTNF-R1 and 2) were determined with ELISA in chronic heart failure patients and age and gender-matched healthy controls.

Results: Serum HER2 was higher (P=0.013) in 50 heart failure patients (18 female; median age 57 (range 33–77) years), mean 12.1±S.D. 2.3 ng/mL, than in 15 controls, 10.4±2.6 ng/mL. Serum HER2 levels correlated inversely with left ventricular ejection fraction (P=0.037) and were highest among NYHA class III patients, followed by NYHA class II patients and controls (P=0.029, Kruskal–Wallis test). STNF-R1 (P<.001) and sTNF-R2 (P=0.015) were higher in patients than controls, and correlated positively with HER2 (P=0.027 and P=0.036, respectively).

Conclusions: Serum HER2 levels are increased in chronic heart failure patients. Further research is necessary to determine whether HER2 plays a role in the pathophysiology of heart failure.

Key Words: HER2 • Heart failure • Cytokine • Cardiotoxicity • Trastuzumab

Received February 5, 2006; Revised April 17, 2006; Accepted May 18, 2006


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
An increased incidence of heart failure is observed with the use of trastuzumab, a monoclonal antibody against the human epidermal growth factor receptor 2 (HER2), also known as erbB-2, in the treatment of HER2-positive breast cancer patients [1,2]. The pathophysiological mechanism of trastuzumab-related heart failure is still unclear. In animals, HER2 is indispensable for normal cardiac development [3]. Mice with a cardiac-restricted HER2 gene deletion developed severe dilated cardiomyopathy, after they were born without overt phenotypic abnormalities and at normal Mendelian frequencies [4,5]. This coincided with increased apoptosis in the left ventricles of these mice [4]. Based on these data, HER2 is considered to play a role in cardiac (patho)physiology.

Circulating levels of the pro-inflammatory cytokines tumor necrosis factor (TNF)-{alpha} and its soluble (s) receptors (R), TNF-R1 and TNF-R2, are elevated in patients with chronic heart failure. Furthermore, these proteins are associated with heart failure severity [6-8] and increased cardiomyocyte apoptosis in these patients [8].

The extracellular domain of the transmembrane HER2 protein can be proteolytically cleaved from the cell membrane. This HER2 extracellular domain can be measured in peripheral blood. Patients with HER2 positive breast cancer have high serum HER2 levels, which correlate positively with the number of metastatic sites [9].

HER2 is hypothesized to be involved in compensatory mechanisms following cardiac stress. Anthracyclines, for instance, can induce cardiac stress through the induction of free oxygen radicals [10], which may lead to upregulation of cardiomyocyte-expressed HER2, rendering these cardiomyocytes more susceptible to trastuzumab. We hypothesize that HER2 is involved in chronic heart failure, reflected by altered serum HER2 concentrations. The aim of the present study was to evaluate circulating HER2 concentrations and whether serum HER2 is associated with plasma levels of the inflammatory cytokines TNF-{alpha}, sTNF-R1 and 2.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Subjects
Consecutive patients who visited the outpatient clinic of the Thorax Center of the University Medical Center Groningen, The Netherlands, for chronic heart failure were asked to participate in the study. Exclusion criteria were unstable cardiac disease, such as decompensated heart failure or unstable angina pectoris, prior anthracycline-based chemotherapy, and serious uncontrolled concurrent disease, or concurrent malignancy.

A control group consisting of gender- and age-matched healthy volunteers was recruited by an advertisement. None of the volunteers were admitted to the hospital, had acute or chronic illness, or reported symptoms related to the cardiovascular system. The investigation conforms with the principles outlined in the Declaration of Helsinki [11].

All subjects gave their written informed consent, in accordance with the approval of the local medical ethics committee.

2.2. Cardiac evaluation
Signs and symptoms of heart failure were classified according to the New York Heart Association (NYHA) functional class. Radionuclide angiography or cardiac ultrasound was performed to determine left ventricular ejection fraction (LVEF). Left ventricular end systolic and diastolic diameter (LVESD and LVEDD) were determined with echocardiography. Peak oxygen consumption was assessed using exercise testing with gas exchange analysis.

2.3. Blood sampling
For the determination of serum HER2, B-type natriuretic peptide (BNP), TNF-{alpha}, sTNF-R1 and sTNF-R2, peripheral blood samples were collected and transferred into 10 mL disposable tubes, containing either 2-natrium-ethylenediamine tetra-acetic acid (EDTA), heparin or no additive. After sampling, tubes were placed on ice immediately. Serum or plasma was separated within 30 min of collection by centrifugation at 4 °C, and stored at –80 °C until determination. HER2 extracellular domain concentrations were measured in serum, BNP and TNF-{alpha} in EDTA plasma. Heparin plasma was used for quantification of sTNF-R1 and sTNF-R2.

2.4. Serum HER2, TNF{alpha}, TNF-Rs and neurohormones
BNP was assessed with an immunoassay (Abbott Laboratories, Abbott Park, IL, USA). Serum HER2 levels were measured using a sandwich enzyme assay (HER-2/ECD Assay, Oncogene Sciences, Cambridge, MA, USA). The assay has a cut-off value of 15 ng/mL, based on serum HER2 levels in breast cancer patients, which correspond with tumor HER2 expression [12]. Plasma TNF-{alpha}, sTNF-R1 and sTNF-R2 were determined using commercially available enzyme-linked immunosorbent assays (Quantikine, R&D systems, Minneapolis, MN, USA) following the manufacturer's instructions.

2.5. Statistics
Values are given as the median (range). Quantitative variables were compared between two groups using a Mann-Whitney U-test for skewed distributed variables. For comparisons between more than two groups, a Kruskal-Wallis test was used. Correlations between variables were calculated using Pearson's correlation coefficient test. All P-values were two-sided and P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Clinical characteristics
Clinical characteristics are presented in Table 1. The study population consisted of 50 patients with moderate to severe chronic heart failure. Medical treatment for heart failure consisted of angiotensin-converting enzyme inhibitors or angiotensin receptor-2 antagonists, β-blockers, diuretics, nitrates and/or digoxin. BNP values were higher in patients, than in controls (P<0.01), and higher in NYHA III than in NYHA II patients (P<0.05) (Table 1). LVEF values correlated inversely with BNP values (R=–0.385, P=0.012) and positively with peak oxygen consumption values (R=0.284, P=0.046). The control group consisted of 15 gender and age-matched healthy subjects (10 men and 5 women) with a median age of 56 (range 39-70) years.


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  		Table 1 Clinical characteristics

 
3.2. Serum HER2, plasma TNF{alpha} and TNFRs
Serum HER2 levels were higher in heart failure patients, compared to controls (P=0.013) (Table 2). Plasma sTNF-RI and sTNF-RII were also higher in patients, than in controls (P<0.001 and P=0.015, respectively). When comparing NYHA class III to NYHA class II and controls, NYHA class III patients had the highest circulating levels of HER2 (P=0.029), sTNF-R1 (P<0.001) and sTNF-R2 (P=0.010), followed by NYHA class II patients and controls (Kruskal-Wallis test) (Fig. 1). Serum HER2 levels were not associated with the time since the first diagnosis of heart failure. No associations were observed between serum HER2 levels and gender, age or the presence of diabetes mellitus, hypertension or present smoking. TNF{alpha} and sTNF-R levels were also not associated with gender, age or any of the aforementioned comorbidities. Serum HER2 correlated inversely with LVEF (R=–0.296, P=0.037). HER2 did not correlate with peak oxygen consumption, plasma BNP values, nor with LVESD or LVEDD. No differences were observed regarding serum HER2, plasma TNF-{alpha} and sTNF-R levels between patients with DCM and IHD as the cause for heart failure. A weak positive correlation existed between serum HER2 and sTNF-R levels; sTNF-R1 (R=0.277, P=0.027), sTNF-R2 (R=0.260, P=0.036).


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  		Table 2 Serum HER2, plasma TNF{alpha} and sTNF-R levels

 


Figure 01
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  		Fig. 1 Dot plot of serum human epidermal growth factor receptor 2 (HER2) (A), tumor necrosis factor receptor (sTNF-R) 1 (B) and sTNF-R2 (C) levels in New York Heart Association (NYHA) class II vs. NYHA class III heart failure patients, compared to controls. Lines represent mean values. P-value based on Kruskal-Wallis test. Dotted line represents the serum HER2 cut-off value (upper limit of normal) of 15 ng/L.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The major finding of the present study is that serum HER2 levels were elevated in chronic heart failure patients due to DCM or IHD. Higher serum HER2 levels were associated with increasing NYHA class, and an inverse correlation between serum HER2 and LVEF was observed. Furthermore, serum HER2 levels correlated positively with plasma levels of the apoptosis-related cytokines sTNF-R1 and sTNF-R2.

However, given the fact that the differences in serum HER2 levels between heart failure patients and controls were relatively small, the clinical relevance of this finding is limited. Nevertheless, the elevated serum HER2 in heart failure patients could suggest that HER2 plays a role in cardiac pathophysiology which may be relevant in the light of the mechanisms responsible for trastuzumab-related cardiac dysfunction. Hypothetically, loss of cardiomyocyte-expressed HER2 may result in loss of functional myocytes leading to functional impairment and heart failure. In our heart failure population, we observed an inverse association between serum HER2 levels and systolic left ventricular function as assessed with LVEF measurement. Serum HER2 was, however, not related to other markers of heart failure severity such as BNP, peak oxygen consumption nor with left ventricular diameters.

Recently, HER2 expression was shown to be present with immunohistochemistry in left ventricle biopsies from 6 of 60 patients with severe cardiac disease and heart failure symptoms. Only weak HER2 staining was observed in these positive biopsies, compared to HER2 overexpressing or HER2 gene amplifying breast tumours [13]. This might explain why circulating HER2 levels in our heart failure patients are only slightly elevated in comparison to high serum HER2 values in HER2 positive breast cancer patients [14].

In rats with banding-induced aortic stenosis, compensatory myocardial hypertrophy precedes heart failure. Myocardial HER2 mRNA expression, which is present during compensatory hypertrophy in the animals with banding-induced aortic stenosis, is decreased during the transition from compensatory hypertrophy to heart failure [15]. Furthermore, HER2 inhibition in isolated neonatal rat ventricular myocytes resulted in increased expression of the pro-apoptotic protein Bcl-xS and a decrease in the anti-apoptotic Bcl-xL [16]. In 36 patients with severe (NYHA class IV) heart failure due to ischaemic or non-ischaemic cardiomyopathy, myocardial HER2 mRNA levels were raised following left ventricular assist device implantation [17]. Rohrbach et al. described recently that both HER2 mRNA and protein were decreased in left ventricular myocardial tissue of explanted hearts of 32 heart failure patients who underwent heart transplantation, compared to 10 donors. Next to an increase in the proapoptotic protein Bcl-Xs, the authors describe that mRNA expression of the soluble form of HER2 was higher in the failing than in donor myocardium [18]. It can thus be imagined that the slightly higher serum HER2, as was observed in our study population, is indicative of increased shedding of HER2 from the cardiomyocyte membrane into the circulation.

Whether or not the myocardium is the origin of the increased HER2 serum levels remains unclear. One possible explanation for the increased serum HER2 levels in heart failure patients is increased shedding of cardiomyocyte membrane-expressed HER2. An alternative explanation for the increased serum HER2 levels may be altered splicing of primary HER2 receptor transcripts. It can also be imagined that the HER2 is produced as an epiphenomenon by other organs, or as a result of systemic responses, such as passive congestion, ischaemia or as an acute phase reactant. HER2 appears to be involved in the prevention of apoptosis [19]. We observed a weak positive correlation between plasma levels HER2 and the pro-apoptotic cytokines TNF-R1 and 2. Despite this correlation, no firm conclusions can be drawn with regard to the role of HER2 in preventing apoptosis, based on the findings of the current study. In addition, only NYHA functional class II-III heart failure patients were enrolled. This indicates that patients' heart failure was well controlled. As a result, true congestion, which might have influenced serum HER2 and cytokine levels, was not observed.

In conclusion, serum HER2 levels in our population of heart failure patients were increased compared controls, which suggests that HER2 plays a role in heart failure. The origin and underlying pathophysiological mechanisms for the higher circulating levels of HER2 remain unclear and require further investigation.


   Acknowledgements
 
We thank F. Hoffmann-La Roche AG and Klinikum Kassel for the measurement of the serum HER2 levels and Abbott for providing us with the BNP radioimmunoassay.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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