Simultaneous expression of c-erbB-1, c-erbB-2, c-erbB-3 and c-erbB-4 receptors in non-small-cell lung carcinomas: Correlation with clinical outcome

Lung Cancer (2007) 57, 193—200

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/lungcan

Simultaneous expression of c-erbB-1, c-erbB-2, c-erbB-3 and c-erbB-4 receptors in non-small-cell lung carcinomas: Correlation with clinical outcome Anastassios V. Koutsopoulos a,∗, Dimitris Mavroudis b, Konstantina I. Dambaki a, John Souglakos b, Eleni G. Tzortzaki c, John Drositis d, George S. Delides a, Vassilis Georgoulias b, Efstathios N. Stathopoulos a a

Department of Pathology, University General Hospital of Heraklion, P.O. Box 1352, Heraklion 71110, Crete, Greece Department of Medical Oncology, University General Hospital of Heraklion, P.O. Box 1352, Heraklion 71110, Crete, Greece c Department of Pulmonary Diseases, University General Hospital of Heraklion, P.O. Box 1352, Heraklion 71110, Crete, Greece d Department of Thoracic Surgery, University General Hospital of Heraklion, P.O. Box 1352, Heraklion 71110, Crete, Greece b

Received 23 November 2006; received in revised form 14 February 2007; accepted 7 March 2007

KEYWORDS Epidermal growth factor receptor family; NSCLC; c-erbB receptors; Lung cancer

∗

Summary The expression of c-erbB receptors was immunohistochemically examined in paraffin embedded specimens from non-small-cell lung carcinomas. A total of 209 patients were enrolled [squamous-cell carcinomas (n = 59), adenocarcinomas (n = 130), large-cell carcinomas (n = 15) and giant-cell carcinomas (n = 5)]. The HercepTest kit scoring guidelines were used for the interpretation of positivity. C-erbB-1 was overexpressed in older patients, in squamouscell carcinomas and in poorly-differentiated tumours, whereas c-erbB-2 overexpression with adenocarcinomas and poorly-differentiated tumours. C-erbB-4 overexpression correlated with advanced disease stage. The c-erbB-1/4 pair was the most commonly overexpressed and significantly correlated with female gender, while the c-erbB-1/2 pair with older age. Response to chemotherapy was significantly reduced in patients with tumours overexpressing c-erbB-1 receptor as well as the c-erbB-1/2 and c-erbB-3/4 receptor pairs. Patients’ overall survival was significantly correlated with the co-expression of c-erbB-1 and c-erbB-4 receptors. These findings clearly suggest that specific receptors overexpression or co-overexpression is correlated with patients’ disease control rate and outcome. A better understanding of the overexpression of the heterodimerized partners of c-erbB family receptors may provide a useful predictive indicator of response to molecular targeted therapies with c-erbB inhibitors. © 2007 Elsevier Ireland Ltd. All rights reserved.

Corresponding author. Tel.: +30 2810394708; fax: +30 2810394694. E-mail address: [email protected] (A.V. Koutsopoulos).

0169-5002/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2007.03.009

194

1. Introduction Homeostasis of the cells depends on diverse stimuli of extracellular matrix and cellular interaction. It is of great importance that these signals are correctly interpreted by the cell in order to achieve an appropriate developmental or proliferative response. Molecular events controlling proliferation in normal and transformed cells involve dominant oncogenes, tumour suppressor genes, cell cycle regulators and growth factor receptors. Growth factors are stimuli that regulate cell proliferation and differentiation through their binding to different transmembrane receptors, some of which show endogenous tyrosine kinase activity. Tyrosine kinase receptors comprise a family of proteins that have been subclassified according to their structural homology. The subgroup of tyrosine kinase receptors type I consists of four members that differ in ligand specificity. The prototype of this subgroup is Epidermal Growth Factor Receptor (EGFR or c-erbB-1), that binds to several different ligands (e.g. TGF-a, Amphiregulin, Betacellulin, HB-EGF and Epiregulin) [1]. C-erbB-3 or Her-3 and c-erbB-4 or Her-4 are two other members, the ligands of which are isomorphs of the new differentiation factor (NDF or neuregulins) [2,3]. The last member of this subgroup is c-erbB-2, also known as c-neu or Her-2, which does not have any known ligand so far [4]. All members consist of an extracellular ligand-binding domain, a single membrane-spanning region and a cytoplasmic protein tyrosine kinase domain. After ligand-receptor binding c-erbB-1, c-erbB-2, c-erbB-3 and c-erbB-4 undergo homo- or heterodimerization and activation of the receptor through autophosphorylation of intracytoplasmic tyrosine kinase domain, which sequentially activates distinct signaling pathways. C-erbB heterodimerization is a means not only for signal amplification but also for signal diversification [1]. Tzahar et al. [5] reported that a hierarchical network of interreceptor interactions determines signal transduction, and suggested that c-erbB-2 is the preferred member of the c-erbB family for heterodimerization. Many studies have reported that c-erbB family and especially the c-erbB-1 and c-erbB-2 receptors are implicated in tumour growth, metastatic capacity and resistance to treatment of many human carcinomas [6,7]. The majority of published data regarding non-small-cell lung carcinoma (NSCLC) are mainly focused on protein and gene expression of c-erbB-1 and c-erbB-2 with conflicting results in terms of protein expression level, gene amplification and influence on survival [8—17]. In the unique study where the expression of all erbB receptors was examined in 73 stage I NSCLCs, cerbB1 and -3 were correlated with poor differentiation and tumour recurrence [18]. Moreover, tyrosine kinase inhibitors have been developed and used in many clinical trials in NSCLCs with equivocal benefit [19—24]. In most of these trials patient selection was not based on the expression of c-erbB protein levels but rather on various clinical criteria. Considering c-erbB receptor homo- and heterodimerization and the fact that some tumours express elevated protein levels for certain c-erbB family members, we decided to examine the expression levels of c-erbB-1, cerbB-2, c-erbB-3 and c-erbB-4, in 209 human NSCLC tissue specimens, by means of immunohistochemistry. The aim of the present study was to determine the expression levels of all c-erbB family receptor members and receptor pairs in

A.V. Koutsopoulos et al. the same tumour sample and to correlate these results with patients’ and tumours’ characteristics as well as patients’ clinical outcome.

2. Material and methods 2.1. Study population Paraffin blocks prepared during the period August 1994 to November 2003 were retrieved from the archives of the Pathology Department of the University General Hospital of Heraklion, Crete, Greece. Cases included patients with NSCLC diagnosed from 91 surgically resected specimens (pneumonectomies, lobectomies) received from the Department of Thoracic Surgery of the University General Hospital of Heraklion, Crete and 118 bronchoscopic biopsies with adequate material performed in the Department of Pulmonary Diseases [25]. All patients were treated at the Department of Medical Oncology. Medical files were reviewed and all relevant clinical (type of treatment, response to chemotherapy, survival, etc.) data were recorded. A total of 209 cases were evaluated immunohistochemically. The study was approved by the Ethics and Scientific Committees of our Institution.

2.2. Immunohistochemistry 2.2.1. Tissue preparation The tissue samples, fixed in 4% neutral buffered formalin overnight and embedded in paraffin, were retrieved from the archives of the Pathology Department. From each representative specimen block, five consecutive 4 ␮m thick sections were cut and were deposited on SuperFrost/Plus Slides (O. Kindler GmbH, Freiburg, Germany). The first one was stained with haematoxylin and eosin (H&E), and reviewed to corroborate the histologic diagnosis and ensure that it was representative of the tumour. The other four slides were used for immunohistochemistry. 2.2.2. Immunostaining The slides were deparaffinized in xylene (after overnight heating at 37 ◦ C and for 1 h at 60 ◦ C), rehydrated in ethanol (treated with a graded series of alcohol 100%, 96%, 80% and 70% ethanol in distilled water) and finally in distilled water. For antigen retrieval, sections analyzed for c-erbB-1 were treated with proteinase K (DakoCytomation, Code S3020) for 5 min at room temperature; sections analyzed for c-erbB-3 and c-erbB-4 were treated with EDTA at pH 8 in a microwave oven three times for 5 min at 500 W and for c-erbB-2 sections were submitted in citric acid monohydrate buffer 0.01 M pH 6, consisting of three times for 5 min of microwave treatments at 500 W. After pretreatment slides were rinsed in distilled water and washed in TBS (three times for 5 min). Primary antibodies used for immunohistochemical staining were mouse monoclonal anti-EGFR antibody (clone H11, code M3563, DakoCytomation, Denmark), mouse monoclonal antibody directed to the internal domain of c-erbB-2 (clone CB11, code NCL CB11, Novocastra Laboratories, UK), rabbit polyclonal antibody against N-terminus of human c-erbB3 protein (Cat #E3434, Spring Bioscience, 46755 Fremont Blvd. Fremont, CA 94538) and rabbit prediluted antibody

Simultaneous expression of c-erbB receptors in non-small-cell lung carcinomas

195

Fig. 1 Synchronous immunohistochemical overexpression of c-erbB-1 and 4 in a 65 years old male patient with squamous-cell carcinoma of the lung (×200).

directed to the C-terminus of human c-erbB-4 protein (Cat #E2761, Spring Bioscience, 46755 Fremont Blvd. Fremont, CA 94538). The working dilutions were for c-erbB-1 1/50, for c-erbB-2 1/50 and for c-erbB-3 1/20, while c-erbB-4 was in a prediluted form. For all of them bovine serum albumin (BSA) solution 30% (Ref HALALB01-OP, Eurobio, France) at a final dilution of 1/100 was used. All the primary antibodies were incubated for 1 h at room temperature. For the detection of antigen-antibody reaction the UltraVision detection system AP Polymer kit (Cat #TL-125-AL, Lab Vision, UK), was used according to the manufacture’s instructions. Fast red was used as chromogen for 20 min; the sections were counterstained with Mayer’s hematoxylin for 3 min, subsequently rinsed in ammonium and finally mounted with glycergel. A positive (breast carcinoma with known positivity) and a negative (omission of primary antibody) control were used for each antibody.

were evaluated with each batch of slides. In the literature there are uniform criteria only for the interpretation of cerbB-2, but the immunoreactivity for the other receptors is not standardized (arbitrary cut-offs and intensities for cytoplasmic or membrane staining). Since all these receptors are located in the cellular membrane, we decided to evaluate the positivity using the classification for c-erbB-2 applied in breast cancer according to the HercepTest kit scoring guidelines approved by the Food and Drug Administration. Immunoreaction was considered as weakly positive (2+) if more than 10% of the tumour cells showed weak to moderate complete membrane staining or as strongly positive (3+) if a strong complete membrane staining was observed in more than 10% of the tumour cells. All other staining patterns were interpreted as negative (0 or 1+). Representative immunohistochemical determinations of c-erbB family members are shown in Figs. 1—3.

2.2.3. Interpretation of immunohistochemistry Two pathologists (K.A.V. and D.K.I.) evaluated the stained sections independently. The results were compared and discordant interpretations were resolved by reviewing of the specific slides by a third pathologist (S.E.N.) under a multihead microscope. A positive and a negative control sample

2.2.4. Statistical methods Samples with a score of 2+ (weak overexpression) or 3+ (strong overexpression) were considered to overexpress that particular protein. The patients’ demographic data (age and sex), tumour type, tumour differentiation, disease stage, medical treatment, response to chemotherapy and overall

Fig. 2 Synchronous immunohistochemical overexpression of c-erbB-2 and 4 in a 63 years old male patient with adenocarcinoma of the lung (×200).

196

A.V. Koutsopoulos et al.

Fig. 3 Synchronous immunohistochemical overexpression of c-erbB-1 and 2 in a 55 years old male patient with adenocarcinoma of the lung (×200).

survival (OS) in crosstabulation with the expression (weak and strong or strong) of the c-erbB proteins were analysed using Pearson’s Chi-square test. The two-sided significance level was set at <0.05. To identify independent predictive factors significantly influencing the incidence of response or death a linear regression analysis was performed. The Kaplan—Meier analysis was used in order to estimate the probability of survival.

C-erbB-1 was overexpressed (2+ and 3+) in 47.7% of tumour samples and this overexpression was correlated with older age (>60 years) (p = 0.003) and with squamous-cell carcinoma histology (p = 0.002). Strong overexpression (3+) was correlated with patients’ age (>60 years) (p = 0.001),

Table 1

Patient characteristics Number

3. Results Patients studied

3.1.1. Patients’ characteristics The studied patients included 189 (90%) males with a median age of 62 years (range 34—90) while 165 (78.95%) had stage IIIB—IV disease. Histological subtypes were recorded according to the WHO classification [26] and most of them were adenocarcinomas (n = 130). Patients’ characteristics are shown in Table 1. The median OS was 12 months (95% CI: 10.0—13.9 months) due to the high percentage of patients with advanced disease. Indeed, patients with stage IV disease had a median OS of 9 months, significantly lower compared with 20 months of patients with stage I—IIIA disease (p < 0.001). One hundred seventy six patients received chemotherapy for advanced disease; the objective response rate was 26% (95% CI: 19—34%).

3.2. Expression of c-erbB family receptors Out of the 209 cases, 199 were evaluable for the expression of all four c-erbB family members. We found c-erbB-1, c-erbB-2, c-erbB-3 and c-erbB-4 overexpression in 47.7% (weakly positive 20.1% and strongly positive 27.6%), 23.7% (weakly positive 12.1% and strongly positive 11.6%), 6.5% (weakly positive 3.5% and strongly positive 3.0%) and 25.2% (weakly positive 12.1% and strongly positive 13.1%) of tumour samples, respectively (Table 2). Approximately 29% of tumour samples were negative for all four c-erbB receptors, while one c-erbB receptor overexpression was found in 43.7%, co-expression of two receptors in 22.6%, coexpression of three receptors in 4.0% and one tumour (0.5%) sample overexpressed all four receptors (Fig. 4).

Age (year) Median Range

%

209 62 34—90

Gender Men Women

189 20

90.4 9.5

Histological type Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Giant cell carcinoma

130 59 15 5

62.2 28.2 7.1 2.3

Tumour differentiation High Moderate Low

14 50 145

6.6 23.9 69.3

Disease stage IA IB IIA IIB IIIA IIIB IV

2 7 6 12 17 59 106

0.9 3.3 2.8 5.7 8.1 28.2 50.7

Prior treatment Surgery Adjuvant chemotherapy Chemoradiotherapy/+-radiotherapy Supportive care

35 14 176 8

16.7 6.7 84.2 3.0

Simultaneous expression of c-erbB receptors in non-small-cell lung carcinomas Table 2

197

C-erbB receptor overexpression and co-overexpression with significant associations

Positivity

% of samples

Age (>60 vs. <60)

Sex (female vs. male)

Tumour type Squamous cell carcinoma

Adenocarcinoma

Tumour differentiation (low vs. high & moderate)

Stage [Advanced (IIIB—IV) vs. early stage]

c-erbB-1 2+ and 3+ 3+

47.7 27.6

p = 0.003 p = 0.001

— —

p = 0.002 p = 0.001

— —

— p = 0.001

— —

c-erbB-2 2+ and 3+ 3+

23.7 11.6

— —

— —

— —

p = 0.013 —

— p = 0.001

— —

c-erbB-3 2+ and 3+ 3+

6.5 3.0

— —

— —

— —

— —

— —

— —

c-erbB-4 2+ and 3+ 3+

25.2 13.1

— —

— —

— —

— —

— —

— p = 0.032

c-erbB-1/2 2+ and 3+ 3+

8.5 2.0

— p = 0.053

— —

— —

— —

— —

— —

c-erbB-1/3 2+ and 3+ 3+

3.5 1.5

— —

— —

— —

— —

— —

— —

c-erbB-1/4 2+ and 3+ 3+

13.1 4.5

— —

— p = 0.004

— —

— —

— —

— —

c-erbB-2/3 2+ and 3+ 3+

1.5 0.5

— —

— —

— —

— —

— —

— —

c-erbB-2/4 2+ and 3+ 3+

7.5 2.5

— —

— —

— —

— —

— —

— —

c-erbB-3/4 2+ and 3+ 3+

3.5 1.5

— —

— —

— —

— —

— —

— —

Fig. 4

Receptor co-expression in NSCLC

squamous-cell carcinoma (p = 0.001) and poorly differentiated tumours (p = 0.001). No correlation was found with sex and clinical stage (Table 2). C-erbB-2 was overexpressed (2+ and 3+) in 23.7% of tumour samples and this overexpression was correlated with adenocarcinoma tumour type (p = 0.013). Strong overexpression (3+) correlated with poorly differentiated tumours (p = 0.001). No significant correlation was found with age, sex and clinical stage (Table 2). C-erbB-3 was overexpressed (2+ and 3+) in 6.5% of tumour samples and this overexpression showed no correlation with age, sex, tumour type, differentiation and clinical stage (Table 2). Besides the membrane positivity, we also noticed a high number of cases expressing nuclear staining. C-erbB-4 was overexpressed (2+ and 3+) in 25.2% of tumour samples and this overexpression was not correlated with age, sex, tumour type or differentiation. Strong overexpression (3+) was significantly correlated with advanced clinical stage (p = 0.032) (Table 2).

198

3.3. Expression of erbB family receptor pairs C-erbB-1/c-erbB-2 pair was co-overexpressed (2+ and 3+) in 8.5% of tumour samples. Strong positivity (3+) for this pair of receptors was found in 2% of samples and was more commonly found in older individuals (>60 years old) (p = 0.053) (Table 2). C-erbB-1/c-erbB-4 pair was co-overexpressed (2+ and 3+) in 13.1% of tumour samples. Strong positivity (3+) for both receptors (4.5%) was significantly correlated with female sex (p = 0.004) (Table 2). The co-overexpression of other pairs was rare (less than 10%) and showed no significant correlation as indicated in Table 2.

3.4. Correlation of expression of erbB family receptors with clinical outcome Strong (3+) overexpression of c-erbB-1 receptor, c-erbB-1/2 and c-erbB-3/4 receptor pairs showed statistically significant correlation with lower response rate to chemotherapy (p = 0.04, p = 0.04 and p = 0.04, respectively). A significant correlation could also be detected between the strong (3+) co-expression of c-erbB-1 and c-erbB-4 receptors and patients’ overall survival (8 months for strong overexpression versus 13.9 months for weak and negative; p = 0.02) (Fig. 5). A marginally significant decreased survival was also observed when c-erbB-3 receptor was strongly (3+) overexpressed (p = 0.05). Overexpression (2+ and 3+) of individual receptors or receptor pairs did not show any significant correlation with disease control rate or median overall survival. Detailed statistical correlations between the strong overexpression of erbB receptors and receptor pairs with disease control rate and median overall survival are shown in Table 3. The multivariate analysis demonstrated that only stage (I—IIIA versus IIIB—IV) of the disease and response to first line treatment (CR+PR versus SD + PD) were independent prognostic factors for overall survival [Hazard Ratio 1.66 (p = 0.01) and 1.62 (p = 0.04), respectively] (Table 4).

A.V. Koutsopoulos et al. Table 3 Correlation of c-erbB receptor overexpression with disease control and overall survival Positivity

DCR (CR + PR + SD)

Overall survival (OS)

%

P-value

Months

P-value

c-erbB-1 Strong Negative

18.6 81.4

0.04

12 13.5

0.95

c-erbB-2 Strong Negative

11.6 88.4

0.87

12 13.5

0.92

c-erbB-3 Strong Negative

3.5 96.5

0.71

10.5 13.9

0.05

c-erbB-4 Strong Negative

9.3 90.7

0.15

14.5 13

0.45

c-erbB-1/2 Strong Negative

0.0 100.0

0.04

12 13.9

0.26

c-erbB-1/3 Strong Negative

2.3 97.7

0.74

12 13.5

0.27

c-erbB-1/4 Strong Negative

1.2 98.8

0.08

8 13.9

0.02

c-erbB-2/3 Strong Negative

0.0 100.0

0.24

12 13.5

0.62

c-erbB-2/4 Strong Negative

3.5 96.5

0.46

14.5 13

0.65

c-erbB-3/4 Strong Negative

0.0 100.0

0.04

12 13.9

0.17

OS, overall survival; DCR, disease control rate; CR, complete response; PR, partial response; SD, stable disease. Strong = 3+ positivity. Statistically significant correletions are presented in bold.

Table 4

Fig. 5 Patients’ overall survival and co-overexpression of cerbB-1/c-erbB4 receptors.

Multivariate analysis for survival

Variable

Hazard ratios

95% CIa

p-Value

Age (>70 vs. ≤70) Sex (male vs. female) Stage (I—IIIA vs. IIIB—IV) Response to 1st line treatment (CR + PR vs. SD + PD) Strong positivityb Any pair positivity

0.94 1.2 1.66 1.62

0.66—1.35 0.63—2.28 1.13—2.46 1.01—2.59

0.77 0.57 0.01 0.04

1.20 1.06

0.82—1.74 0.69—1.63

0.33 0.78

Statistically significant correletions are presented in bold. a Confidence interval. b To any of the erbB family receptor member.

Simultaneous expression of c-erbB receptors in non-small-cell lung carcinomas

4. Discussion The protein expression levels of all c-erbB family members were evaluated in the present study by immunohistochemistry in 209 human NSCLC tissue specimens. It was observed that c-erbB-1 receptor protein was overexpressed in 47.7% of tumour samples and this positivity was correlated with older age (>60 years), squamous-cell carcinoma histology and poorly differentiated tumours. These observations are in accordance with previous studies [9,11,12,15,17,18,27—29], although other studies have failed to demonstrate a clear correlation between these parameters [11,14,17,30]. CerbB-2 receptor protein was overexpressed in 23.7% of tumour samples and this overexpression correlated with an adenocarcinoma subtype. Some previous studies are in agreement with our results [12,30], while others are not [8,14,27,31]. Strong overexpression (3+) correlated with poorly differentiated tumours. C-erbB-3 and c-erbB-4 were overexpressed in 6.5% and 25.2% of tumour samples, respectively. C-erbB-4 overexpression was strongly correlated with an advanced clinical stage. Our results concerning c-erbB-3 expression level differ from those reported by Hilbe et al. [32] who found 58% protein expression for c-erbB-3, in cryostat sections and those by Lai et al. [18] who reported 64% positivity including cytoplasmic and membrane staining. Obviously discrepancies in c-erbB protein expression levels reported in various immunohistochemically studies of NSCLC could be attributed to the different types of specimen fixation, the antibodies used and the different levels that defined overexpression. Furthermore, some recent studies have pointed out the nuclear staining for c-erbB-1 and cerbB-4 protein and its significance (possible transcriptional factor activity) in breast carcinomas [33,34]. Nuclear positivity of c-erbB-3 was observed in our study in some of the examined specimens. However, since only membrane positivity was considered in the present study, further investigation is needed to evaluate the biological significance of nuclear staining. Demonstration of c-erbB overexpression in NSCLC using a standardized method is essential for clinical trials utilizing anti-c-erbB targeted agents. Taking into account the heterodimerization of the c-erbB receptors, the expression levels of the six possible receptor pairs was examined. It was hypothesized that the synchronous overexpression of c-erbB member pairs in NSCLC tumours might indicate heterodimerization, thus leading to enhanced proliferation capacity of the tumour. In addition, different patterns of heterodimerization may produce different biological effects in terms of pathways activation, as previously proposed [5]. This could influence the biological characteristics of the tumour and affect the prognosis of the patients. The c-erbB-1/4 pair was the most commonly overexpressed (13.1%) and strong (3+) positivity of this pair was significantly correlated with female sex. Whether this observation may be associated with the higher probability of response of female patients treated with tyrosine kinase inhibitors of EGFR [24] needs further investigation. Furthermore, the c-erbB-1/2 receptor pair was overexpressed in 8.5% of tumour samples and strong positivity (3+, 2%) showed a trend to older patients (>60 years old). Onn et al. [12] found synchronous overexpression of c-erbB-1 and c-erbB-2 in 9.9% of the tumour samples, whereas a slightly higher percentage (11%) was found by Meert et al. [14]. The formation

199

of constitutive c-erbB-1/2 heterodimers has also been found in the human NSCLC cell line A549 [35]. The remaining four pairs c-erbB-1/3, c-erbB-2/3, c-erbB-2/4 and c-erbB-3/4 were co-expressed in 3.5, 1.5, 7.5 and 3.5%, respectively, but without any significant correlation with age, sex, tumour type, differentiation and clinical stage. Those findings support the development and use of small molecules that inhibit tyrosine kinases of multiple c-erbB family receptors. The simultaneous inhibition of several cerbB receptors might enhance the anti-tumour activity of these drugs [36]. In the current study, the expression of c-erbB1 receptor, c-erbB-1/c-erbB-2 and c-erbB-3/c-erbB-4 receptor pairs was associated with a lower disease control rate. In addition, a decreased median overall survival was observed in patients whose tumours overexpressed c-erbB-3 receptor and co-overexpressed c-erbB-1/c-erbB-4 receptors. The multivariate analysis failed to demonstrate a significant correlation of strong receptors expression or any pair coexpression as independent prognostic factors for overall survival. Some reports suggest a possible prognostic and predictive value for the expression of one of the erbB receptors [9,10,12—14,16—18]. On the other hand, other studies do not support this suggestion and the issue is still unclear [8,11,15].

5. Conclusion Although further studies are needed to assess the association between receptor expression and clinicopathological parameters and survival, our findings clearly suggest that specific receptors overexpression or co-overexpression is correlated with patients’ disease control rate and outcome. Moreover, it is important to elucidate the precise role of erbB family receptors expression or co-expression, especially in patients treated with novel agents that target these proteins, in order to provide useful predictive indicators of response to molecular targeted therapies with c-erbB inhibitors.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements The authors wish to acknowledge Mrs. Karidi Elli for expert technical assistance. This study was supported by grants from the Cretan Association for Biomedical Research (C.A.B.R.) and the Hellenic Oncology Research Group (H.O.R.G.).

References [1] Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159—67. [2] Peles E, Yarden Y. Neu and its ligands: from an oncogene to neural factors. Bioessays 1993;15:815—24. [3] Chen X, Levkowitz G, Tzahar E, Karunagaran D, Lavi S, BenBaruch N, et al. An immunological approach reveals biological

200

[4]

[5]

[6] [7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

A.V. Koutsopoulos et al. differences between the two NDF/heregulin receptors. ErbB-3 and ErbB-4. J Biol Chem 1996;271:7620—9. Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus-Porta D, Ratzkin BJ, et al. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 1996;15:254—64. Tzahar E, Waterman H, Chen X, Levkowitz G, Karunagaran D, Lavi S, et al. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 1996;16:5276—87. Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37(Suppl. 4):S9—15. Tzahar E, Yarden Y. The ErbB-2/HER2 oncogenic receptor of adenocarcinomas: from orphanhood to multiple stromal ligands. Biochim Biophys Acta 1998;1377:M25—37. Moldvay J, Scheid P, Wild P, Nabil K, Siat J, Borrelly J, et al. Predictive survival markers in patients with surgically resected non-small cell lung carcinoma. Clin Cancer Res 2000;6:1125—34. Cox G, Jones JL, O’Byrne KJ. Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable nonsmall cell lung cancer. Clin Cancer Res 2000;6:2349—55. Ohsaki Y, Tanno S, Toyoshima E, Fujiuchi S, Nishigaki Y, et al. Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol Rep 2000;7:603—7. Hirsch FR, Varella-Garcia M, Bunn Jr PA, Di Maria MV, Veve R, Bremmes RM, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003;21:3798—807. Onn A, Correa AM, Gilcrease M, Isobe T, Massarelli E, Bucana CD, et al. Synchronous overexpression of epidermal growth factor receptor and HER2-neu protein is a predictor of poor outcome in patients with stage I non-small cell lung cancer. Clin Cancer Res 2004;10:136—43. Meert AP, Martin B, Paesmans M, Berghmans T, Mascaux C, Verdebout JM, et al. The role of HER-2/neu expression on the survival of patients with lung cancer: a systematic review of the literature. Br J Cancer 2003;89:959—65. Meert AP, Martin B, Verdebout JM, Noel S, Ninane V, Sculier JP. Is there a relationship between c-erbB-1 and c-erbB-2 amplification and protein overexpression in NSCLC? Lung Cancer 2005;47:325—36. Meert AP, Martin B, Delmotte P, Berghmans T, Lafitte JJ, Mascaux C, et al. The role of EGF-R expression on patient survival in lung cancer: a systematic review with meta-analysis. Eur Respir J 2002;20:975—81. Kern JA, Slebos RJ, Top B, Rodenhuis S, Lager D, Robinson RA, et al. C-erbB-2 expression and codon 12 K-ras mutations both predict shortened survival for patients with pulmonary adenocarcinomas. J Clin Invest 1994;93:516—20. Selvaggi G, Novello S, Torri V, Leonardo E, De Giuli P, Borasio P, et al. Epidermal growth factor receptor overexpression correlates with a poor prognosis in completely resected nonsmall-cell lung cancer. Ann Oncol 2004;15:28—32. Lai WW, Chen FF, Wu MH, Chow NH, Su WC, Ma MC, et al. Immunohistochemical analysis of epidermal growth factor receptor family members in stage I non-small cell lung cancer. Ann Thorac Surg 2001;72:1868—76. Fukuoka M, Yano S, Giaccone G, Tamura T, Nakagawa K, Douillard JY, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced nonsmall-cell lung cancer (The IDEAL 1 Trial) [corrected]. J Clin Oncol 2003;21:2237—46.

[20] Herbst RS, Giaccone G, Schiller JH, Natale RB, Miller V, Manegold C, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial—–INTACT 2. J Clin Oncol 2004;22:785—94. [21] Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial—–INTACT 1. J Clin Oncol 2004;22:777—84. [22] Shepherd FA, Rodrigues PJ, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005;353:123—32. [23] Herbst RS, Prager D, Hermann R, Fehrenbacher L, Johnson BE, Sandler A, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005;23:5892—9. [24] Brown ER, Shepherd FA. Erlotinib in the treatment of non-small cell lung cancer. Expert Rev Anticancer Ther 2005;5:767—75. [25] Meert AP, Martin B, Verdebout JM, Paesmans M, Berghmans T, Ninane V, et al. Correlation of different markers (p53, EGF-R, c-erbB-2, Ki-67) expression in the diagnostic biopsies and the corresponding resected tumors in non-small cell lung cancer. Lung Cancer 2004;44:295—301. [26] Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of tumours of lung and pleura. In: Sobin LH, editor. World Health Organization International Classification of Tumors. 3rd ed. Springer Verlag; 1999. [27] Cox G, Jones JL, Andi A, Waller DA, O’Byrne KJ. A biological staging model for operable non-small cell lung cancer. Thorax 2001;56:561—6. [28] Hirsch FR, Scagliotti GV, Langer CJ, Varella-Garcia M, Franklin WA. Epidermal growth factor family of receptors in preneoplasia and lung cancer: perspectives for targeted therapies. Lung Cancer 2003;41(Suppl. 1):S29—42. [29] Reissmann PT, Koga H, Figlin RA, Holmes EC, Slamon DJ. Amplification and overexpression of the cyclin D1 and epidermal growth factor receptor genes in non-small-cell lung cancer. Lung Cancer Study Group. J Cancer Res Clin Oncol 1999;125:61—70. [30] Reinmuth N, Brandt B, Kunze WP, Junker K, Thomas M, Achatzy R, et al. Ploidy, expression of erbB1, erbB2, P53 and amplification of erbB1, erbB2 and erbB3 in non-small cell lung cancer. Eur Respir J 2000;16:991—6. [31] Selvaggi G, Scagliotti GV, Torri V, Novello S, Leonardo E, Cappia S, et al. HER-2/neu overexpression in patients with radically resected nonsmall cell lung carcinoma. Impact on long-term survival. Cancer 2002;94:2669—74. [32] Hilbe W, Dirnhofer S, Oberwasserlechner F, Eisterer W, Ammann K, Schmid T, et al. Immunohistochemical typing of non-small cell lung cancer on cryostat sections: correlation with clinical parameters and prognosis. J Clin Pathol 2003;56:736—41. [33] Lo HW, Xia W, Wei Y, Ali-Seyed M, Huang SF, Hung MC. Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer. Cancer Res 2005;65:338—48. [34] Srinivasan R, Gillett CE, Barnes DM, Gullick WJ. Nuclear expression of the c-erbB-4/HER-4 growth factor receptor in invasive breast cancers. Cancer Res 2000;60:1483—7. [35] Nakamura H, Takamori S, Fujii T, Ono M, Yamana H, Kuwano M, et al. Cooperative cell-growth inhibition by combination treatment with ZD1839 (Iressa) and trastuzumab (Herceptin) in non-small-cell lung cancer. Cancer Lett 2005;230: 33—46. [36] Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. Lapatinib plus capecitabine for HER2positive advanced breast cancer. N Engl J Med 2006;355: 2733—43.