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Clinical Cancer Research 13, 2998-3005, May 15, 2007. doi: 10.1158/1078-0432.CCR-06-2432
© 2007 American Association for Cancer Research
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Cancer Therapy: Clinical

Epidermal Growth Factor Receptor Expression in High-Grade Osteosarcomas Is Associated with a Good Clinical Outcome

Christian Kersting1, Carsten Gebert2, Konstantin Agelopoulos1, Hartmut Schmidt1, Paul J. van Diest4, Heribert Juergens3, Winfried Winkelmann2, Matthias Kevric6, Georg Gosheger2, Burkhard Brandt5, Stefan Bielack3,6 and Horst Buerger1,7

Authors' Affiliations: 1 Institute of Pathology and 2 Department of Orthopedic Surgery, University of Münster; 3 Department of Pediatric Hematology and Oncology, University Children's Hospital Münster, Münster, Germany; 4 Department of Pathology, University of Utrecht, Utrecht, the Netherlands; 5 Institute of Tumour Biology, University of Hamburg, Hamburg, Germany; and 6 Olgahospital Stuttgart, Pediatrics 5 (Oncology, Hematology, Immunology), and 7 Institute of Pathology, Paderborn, Germany

Requests for reprints: Horst Buerger, Institute of Pathology, University of Münster, Albert-Schweitzer-Strasse 33, 48149 Münster, Germany. Phone: 49-2-51-83-55450; Fax: 49-2-51-83-55460; E-mail: buerger{at}histopatho.eu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The expression of the epidermal growth factor receptor (EGFR) in osteosarcomas has repeatedly been described. With the introduction of anti-EGFR–targeted therapies in clinical practice, these findings regain increased attention. Experience with anti-EGFR–targeted therapies in other cancers has made clear that besides the expression status of EGFR, a detailed knowledge about gene mutations is of major predictive power. We therefore aimed to explore the EGFR expression and gene mutation status in high-grade osteosarcomas.

Experimental Design: We investigated tumor samples of osteosarcoma patients of all age groups by means of immunohistochemistry (n = 111) and egfr fluorescence in situ hybridization (n = 39). Sixty-three patients were treated according to the Cooperative Osteosarcoma Study Group protocols and complete clinical follow-up was available in these cases.

Results: Ninety-one of 111 (81%) of osteosarcomas revealed an expression of EGFR. EGFR expression showed a dose-response relation with improved event-free and overall survival. This was independent of the degree of tumor regression due to neoadjuvant chemotherapy. Nine of 39 (23%) osteosarcomas showed egfr amplifications by means of fluorescence in situ hybridization. All these cases expressed EGFR. When comparing EGFR expression between primary biopsy and resection specimen (n = 19), viable residual tumor cells in resection specimens revealed a lower EGFR expression and a tendency toward membranous staining compared with the initial biopsy.

Conclusions: In conclusion, expression and amplification of EGFR are frequently observed in high-grade osteosarcomas and are associated with improved prognosis in a dose-responsive way. This implies that low EGFR expression possibly predicts lack of response to conventional treatment in high-grade osteosarcomas and may warrant a more intensive therapeutic approach, although not based on EGFR targeting.

The erbB family receptor tyrosine kinases play an important role in the control of cell cycle, proliferation, and migration of normal and cancer cells. Expression of erbB members is linked to poor prognosis in numerous epithelial and mesenchymal cancers (1–3). Therefore, expression analysis of these markers proves useful for evaluation of prognosis in patients suffering numerous different malignancies. Unfortunately, for osteosarcoma patients, only very limited prognostic or predictive markers are available today. Expression of erbB molecules, especially the epidermal growth factor receptor (EGFR), has been repeatedly described in osteosarcomas. ErbB2 expression has even been proved to predict for poor outcome in osteosarcoma patients (4). As of yet, correlation of EGFR expression, related mutations, and clinical outcome has not been drawn (5–7). Although the treatment modalities and the success rate in the treatment of high-grade osteosarcomas have significantly improved over the last decades (4, 8), therapeutic options for advanced disease are rather limited (9). Despite the mentioned advances and although in many cases an initial complete remission can be reached after multimodal therapy, still 30% to 40% of all patients with high-grade osteosarcomas die of metastases or locally recurrent disease. Targeted therapies based on molecular knowledge of critical genetic events may offer new possibilities for patients with advanced disease (10). These therapies aim to interfere with intracellular pathways regulated by membranous tyrosine kinase receptors such as c-erbB2 (breast cancer), c-kit (gastrointestinal stromal tumors), or EGFR (lung cancer). The focus on these molecules as potential therapy targets is further justified by the fact that protein expression in these respective tumors is associated with a significantly poorer prognosis.

In analogy to these considerations but despite the lack of detailed knowledge of egfr related underlying genetic changes and their interaction with tumor proliferation and spread, the application of viral vector–based, anti-EGFR–directed treatment strategies in the treatment of osteosarcomas has been proposed (5).

To achieve deeper insight into the role of EGFR in osteosarcomas and to investigate its potential prognostic value, we aimed to define the role of EGFR in tumor biology of high-grade central osteosarcomas. Therefore, we evaluated the frequency of EGFR expression as well as of egfr amplifications in high-grade osteosarcomas by means of immunohistochemistry and fluorescence in situ hybridization (FISH) to assess whole gene amplifications. Based on the presented results, we show that an improved understanding of the biological role of egfr in osteosarcomas is mandatory before anti-EGFR treatment strategies can be introduced into clinical practice for these tumors.


   Materials and Methods
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 Materials and Methods
Results
 Discussion
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Patients and tumor samples. Formalin-fixed and paraffin-embedded biopsy specimens, originating from 111 non-pretreated high-grade (according to WHO) osteosarcoma patients, were analyzed retrospectively. There were 65 males and 46 females with an average age of 29 years (range, 0-88 years).

The patients were biopsied and treated in the University Hospital of Münster, Germany in the time period between 1990 and 2000. Sixty-three patients received preoperative and postoperative chemotherapy according to the appropriate Cooperative Osteosarcoma Study Group (COSS) protocols (reviewed and approved by the appropriate ethics committee) after having given informed consent (Table 1 ).


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  		Table 1. Clinicopathologic details of 111 osteosarcoma patients and 62 osteosarcoma patients treated with COSS protocols

 
All protocols included methotrexate (12 g/m2) with leucovorin rescue, cisplatin (90-150 mg/m2), doxorubicin (60-90 mg/m2), and ifosfamide (6-10 g/m2). The scheduled duration of chemotherapy ranged from 24 to 38 weeks. Definite surgery was scheduled to take place between weeks 9 and 11. Follow-up was started at time of diagnosis and was done as previously described (8).

Postchemotherapeutic tumor regression was determined using standard procedures (11). A good response to chemotherapy was defined as <10% viable residual tumor cells. Fifty-three percent of all patients showed a good response.

Immunohistochemistry. Slides of the 111 primary initial biopsy samples were deparaffinized and rehydrated. The same was done with 19 resection specimens with good and poor chemotherapeutic response and intermediate to strong EGFR expression in the initial biopsy. After treatment with I-View Inhibitor at 42°C for 4 min, antigen retrieval was done with proteinase K for 6 min in 42°C. The slides were then incubated with the primary antibody (Ventana clone 3C6, prediluted) for 32 min in 42°C in the Ventana Benchmark device. The antibody was chosen after extensive validation on soft tissue sarcoma (12) and breast cancer tissues,8 revealing high sensitivity. After washing, signal amplification was done by incubation with I-View Biotin Ig and I-View SA-HRP for 8 min each at 42°C. The signal was then visualized with I-View DAB and I-View H2O2 for 8 min and enhanced by incubation with I-View COPPER for 4 min. Finally, the slides were counterstained with hematoxylin. Washing steps were included after every incubation.

Because the immunohistochemical EGFR staining patterns in the primary biopsies made a clear distinction between membranous and cytoplasmic staining almost impossible, EGFR membranous and cytoplasmic expressions of tumor cells were scored together. Therefore, for evaluation of EGFR expression, membranous and cytoplasmic staining of tumor cells was scored from 0 to 3 [0, no staining; 1, weak (at least 10% cells with a faint staining intensity); 2, moderate (at least 10% cells with a moderate staining intensity); 3, strong (at least 10% cells with a strong staining intensity)]. This scoring was done by two independent observers (C.K. and H.B.) without knowledge of clinical outcome or other tumor biological features. Discrepant cases were decided on consensus.

FISH. Thirty-nine samples of the series of 111 osteosarcoma samples were successfully analyzed by FISH. The smaller number was due to technical reasons. The probe for egfr detection was derived from a Homo sapiens PAC clone containing the whole egfr gene (GenBank accession no. AC006977). Nick translation was done following standard protocols for labeling of DNA with digoxigenin-11-dUTP. For denaturation, the probe was applied for 5 min at 70°C to 70% formamide-0.6x SSC. Slides underwent pretreatment with proteinase K for 45 min in 45°C. Hybridization to tissue microarray sections of 4-µm thickness was carried out overnight at 37°C in a 50% formamide-1x SSC-10% dextran sulfate solution in the presence of Cot-1-DNA (Life Technologies, Inc.) and HPL-DNA (Sigma). Posthybridization washes were done at 45°C in 50% formamide-2x SSC and 0.1x SSC at 60°C followed by blocking with 3% bovine serum albumin in 4x SSC at 37°C. Probes were detected using mouse anti-digoxigenin (Sigma) and Cy3-labeled goat anti-mouse antibodies (Dianova GmbH) for 45 min each at 37°C (13).

For each tumor biopsy sample, 20 nuclei were selected for scoring according to morphologic criteria using 4',6-diamidino-2-phenylindole counterstaining. Only nonoverlapping, intact nuclei were scored. Clearly distinguishable nontumor cells were disregarded. The cutoff for considering a case as amplified was defined as 4 signals per nucleus. Scoring was done as previously published (14, 15).

Statistical analysis Event-free and overall survival curves were calculated using the Kaplan-Meier method for the COSS-treated patients (16). The comparisons between different groups were based on log-rank testing and multivariate Cox regression analysis. All P values were two sided; P < 0.05 was considered significant. A correlation analysis between patient age, neoadjuvant chemotherapy response, and EGFR expression was done with frequency tables.

All calculations were done with the SPSS software package (SPSS).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
EGFR expression. Twenty-one of 111 tumor samples were negative for EGFR and 30 samples revealed a weak (1+) intracytoplasmic expression of EGFR. Forty-two specimens revealed an intermediate (2+) and 18 specimens a strong (3+) expression of EGFR. No difference in EGFR expression was observed between patient groups above or below 40 years. Comparable frequencies of EGFR expression were observed in all histologic subgroups of osteosarcomas. Examples of immunohistochemical stainings are given in Figs. 1 and 2 .


Figure 1
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  		Fig. 1. A to D, examples of immunohistochemical stainings for EGFR in four osteosarcoma samples with increasing intensity. The expression was seen in the cytoplasma and the cellular membrane. E and F, two osteosarcoma samples without and with amplification of the whole egfr gene, respectively.

 

Figure 2
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  		Fig. 2. Two comparative examples of EGFR expression in osteosarcomas. An initial biopsy (left) and the respective resection specimen with viable tumor cells (right). A tendency toward a decreased and membranously accentuated EGFR expression in the resection specimen is obvious.

 
In the 63 patients treated according to the COSS protocols, EGFR expression was significantly correlated with improved overall and event-free survival. Decreasing EGFR expression was paralleled by decreasing survival, indicating a dose-response relation between EGFR expression and prognosis (Fig. 3 ).


Figure 3
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  		Fig. 3. Kaplan-Meier survival curves for 62 patients with high-grade central osteosarcoma. A and B, curves indicate the overall survival (OS) with regard to an initial complete response and the presence or absence of primary metastasis. C and D, curves show the event-free survival (EFS) and overall survival, respectively, in osteosarcoma patients according to the EGFR expression status. The P values are indicated.

 
In further analysis, tumors with complete lack of EGFR expression were compared with tumors with any (weak, moderate, or strong) EGFR expression. Then, tumors with weak expression or lacking expression were compared with tumors with moderate or strong expression. As shown in Figs. 4 and 5 , the strongest correlation with poor survival could be observed in osteosarcomas lacking EGFR expression. This association became even more evident when excluding patients with initial distant metastases or with incomplete surgical remission after initial therapy. None of the 10 patients with strong EGFR expression died of disease during the observation period, although even one patient presented with initial metastasis. This finding was irrespective of the histologic subtype; all of these patients had a Salzer-Kuntschik score <5.


Figure 4
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  		Fig. 4. Kaplan-Meier curves in 62 patients with osteosarcoma. Patients were grouped according to absent EGFR expression (0) in contrast to all other groups with any kind of EGFR expression (rest). The event-free and overall survival curves are indicated for all patients (A and B), for patients with an initial complete response (C and D), and for patients with or without primary metastasis (E and F). The P values are given. The highest significances became obvious in the overall survival.

 

Figure 5
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  		Fig. 5. Kaplan-Meier curves in 63 patients with osteosarcoma. Patients were grouped according to EGFR expression absent or weak (0/1) in contrast to patients with intermediate or strong EGFR expression (2/3). Event-free and overall survival curves for all patients (A and B), for patients with an initial complete response (C and D), and for patients with or without primary metastasis (E and F). The P values are given. Again, the highest significances became obvious in the overall survival.

 
In 19 cases the immunohistochemical staining pattern for EGFR expression could be compared between the initial prechemotherapeutic biopsy and the resection specimen in cases with incomplete tumor regression. A trend toward a lower and membranously accentuated EGFR expression level could be observed in the viable osteosarcoma cells in the resection specimen. Examples of staining results are shown in Fig. 2.

FISH analysis. Thirty-nine tumor samples covering the complete range of EGFR expression were analyzed by means of FISH. Nine (23.1%) cases displayed whole gene amplifications of egfr (Fig. 1). In seven of these cases, an additional FISH analysis for centromere 7 was successfully done in a serial tumor section. In only one case a significantly higher number of signals for egfr compared with centromere 7 could be observed (centromere 7: 5.05 signals per nucleus compared with egfr: >10 signals). This points toward a predominant polyploidy (chromosome 7 polysomy) in osteosarcomas.

Clinicopathologic correlations. Only trends toward statistical significance could be observed for the relationship between EGFR expression and whole gene egfr amplifications with the tumor regression status after primary neoadjuvant chemotherapy (P = 0.07 and P = 0.11, respectively). A correlation between EGFR expression and tumor site and/or histologic subtype was not observed.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Expression of EGFR has been described in primary osteosarcomas and osteosarcoma cell lines in a rather high frequency (6, 7). Therefore, EGFR is considered to be an attractive target for anti-EGFR–directed, therapeutic approaches, as earlier proposed for c-erbB2 (17). However, up to now, correlations between EGFR expression and clinical outcome have not been published. In accordance with other recent studies (5, 6), we observed the expression of EGFR to be a frequent finding, present in the majority of osteosarcomas, irrespective of histologic subtype, patient age, or tumor site. Interestingly, expression of EGFR in high-grade osteosarcoma showed a positive dose-response relation with favorable clinical outcome. In striking contrast to various epithelial tumor entities (1, 2), tumors with lack or a very weak expression of EGFR had the poorest prognosis. Noteworthy, all patients with strong EGFR expression were alive after up to 12 years of follow-up (overall survival), irrespective of the degree of neoadjuvant chemotherapy–induced tumor regression. The correlation between EGFR expression and outcome became even more striking when patients with initial metastases and patients who never reached a complete remission after initial multimodal therapy were excluded from the analysis. In this selected subgroup, highly significant differences were obtained. It has to be stressed that the results were independent of the scoring methods used. The fact that clear differences could be seen between osteosarcomas completely lacking EGFR expression and all other tumors makes an immunohistochemical interpretation bias (often inherent to semiquantitative evaluations) unlikely.

Interpretation of our findings is not easy. Although the observed relation between EGFR expression and prognosis may merely be a statistical association, the protein dosage effect points to a functional relation. This hypothesis is further underlined by the significantly lower expression in the resection specimens compared with the paired initial biopsy, pointing to selection of lower EGFR-expressing, putatively less chemosensitive tumor cells during the chemotherapy phase.

Interestingly, some literature data from other tumor and nontumor tissues give at least some evidence for the proposed functional relationship. The level of EGFR expression or the activation of the EGFR pathway was associated with an increased sensitivity to topoisomerase I inhibitors and cisplatin in squamous cell carcinoma cell lines (3). Additional hints for a relationship between EGFR expression and an increased sensitivity to cisplatin have further been described in renal tubular cells (18). A similar influence on patient prognosis regardless of postchemotherapeutic tumor regression has been shown repeatedly for P-glycoprotein (19, 20). A further potential link between EGFR expression and chemosensitivity lies in the fact that osteoblastic precursor cells, which in general are more chemosensitive than stem cells, show physiologic EGFR expression (21, 22).

Irrespective of the underlying biological mechanisms, these findings raise important questions about the application of anti-EGFR–directed therapies in osteosarcoma patients. Whereas EGFR expression indicates a poor survival in many tumor entities, one cannot rule out that a blockage of EGFR signaling in osteosarcomas might be associated with an adverse effect (23). Noteworthy, an unexpected inhibition of P-glycoprotein by EGFR-inhibiting agents has recently been described (24, 25).

In recent studies, it could be shown that for predictive purposes, inherited or underlying egfr mutations might be of higher importance than the mere EGFR expression status. Whereas in non–small-cell lung carcinoma, mutations in the intracellular domain of egfr were found to be of predictive value (26), the length of a polymorphism in intron 1 of egfr seems to be of higher importance in squamous cell carcinomas (27). Our results show that the expression of EGFR in osteosarcomas is not obligatorily caused by nor associated with whole gene amplification. Whole gene amplifications of egfr were seen in the present study in slightly more than 20% of all analyzed tumors. However, it seems that most of these amplifications were due to chromosome 17 polysomy, and true egfr amplifications were rather an exception. Nevertheless, egfr-amplified osteosarcomas were consistently EGFR positive. The low frequency of true egfr amplification is in line with recent studies using comparative genomic hybridization or chromosome banding techniques (28–31). Other thus far undescribed egfr mutations might account for EGFR overexpression in osteosarcomas. Alternatively, the expression of EGFR may be regulated on the transcriptional level via p53 (32). Another interesting point is the striking difference from other studies in EGFR as well as other erbB receptors with respect to prognostic or predictive value. Usually, EGFR as well as erbB2 is linked to unfavorable prognosis in numerous epithelial and mesenchymal malignancies. For erbB2, Gorlick et al. (4) even showed correlation with chemotherapeutic tumor response necrosis, suggesting effectiveness via an increased chemosensitivity. Both observations cannot be confirmed for EGFR: in our study, EGFR expression correlated with good prognosis and was not related to chemotherapy induced necrosis. Up to now, there is no sufficient explanation for this unexpected finding. It is therefore obvious that a very delicate interplay, probably mediated via a multitude of different heterodimers of members of the erbB gene family such as c-erbB2, seems to exist, explaining the contradictory roles of EGFR and c-erbB2 in osteosarcomas (33).

In summary, our results give for the first time a detailed insight into EGFR expression patterns in high-grade osteosarcomas and its influence on patient survival, an improved understanding of which could enhance the efficacy of conventional chemotherapeutic treatment regimens. In view of the observed positive relation between EGFR expression and patient survival, high-grade osteosarcomas are unlikely candidates for EGFR-targeted therapy.


   Footnotes
 
Grant support: Peter-and-Traudl-Engelhornstiftung (K. Agelopoulos), Innovative Medizinische Forschung Münster grant KE 2-2-0507, Cancer Institute New York (H. Buerger), EuroBoNet, KP 6 Network of Excellence contract no. 018814, and Deutsche Krebshilfe.

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.

Note: C. Kersting and C. Gebert contributed equally to this work.

8 Unpublished results. Back

Received 10/ 4/06; revised 12/22/06; accepted 1/ 5/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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