Epidermal growth factor receptor (EGFR) status in primary colorectal tumors correlates with EGFR expression in related metastatic sites: biological and clinical implications

A. Italiano1,*, M.-C. Saint-Paul2, F.-X. Caroli-Bosc3, E. François1, A. Bourgeon3, D. Benchimol3, J. Gugenheim3 and J.-F. Michiels2

1 Centre Regional de Lutte Contre le Cancer Antoine-Lacassagne, Department of Medical Oncology, Nice France 2 Centre Hospitalier Universitaire de Nice, Hôpital Pasteur, Department of Pathology, Nice France 3 Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Medical-Surgical Department of Gastrointestinal Oncology and Hepatology, Nice, France

* Correspondence to: Dr A. Italiano, 5 avenue Philippe Rochat, 06600 Antibes, France. Tel: +33-04-89-89-00-60; Fax: +33-04-92-03-10-46; Email: antoineitaliano{at}cario.fr


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of colorectal cancer (CRC). There are several potential strategies to target EGFR. However, monoclonal antibodies and low molecular weight tyrosine kinase inhibitors are the most advanced in clinical development. The majority of studies so far have merely required EGFRs to be expressed by CRC cells. The detection of EGFR expression is usually performed by immunohistochemistry (IHC) in the primary tumor. There are few data regarding the EGFR status in the corresponding distant metastases.

Patients and methods: EGFR status was analyzed by IHC in 80 patients (31 male, 49 female) with CRC (70 colon, 10 rectum) and at least one distant metastatic lesion. Metastatic sites analyzed (n=80) were liver (79 patients) and lung (one patient).

Results: EGFR reactivity was similar in the primary tumor and the related metastases. Among the 80 paired primary/metastatic tumors, only five (6.3%) showed discordance in EGFR status: two cases with EGFR expression in the primary tumor but not in the metastasis, and three samples with EGFR expression in the metastasis but not in the primary tumor.

Conclusions: Between the paired primary tumors and distant metastatic lesions, 94% of samples had concordant EGFR status when analyzed by IHC.

Key words: colorectal cancer, epidermal growth factor receptor, targeted therapies


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that belongs to the ErbB family and is abnormally expressed and activated in cancer cells in many tumor types. Following stimulation by its natural ligands, EGFR initiates signal transduction cascades that promote cell division, migration and angiogenesis, and inhibit apoptosis.

There is clear evidence that EGFR plays an important role in the pathogenesis of colorectal carcinoma (CRC). EGFR is widely present in advanced CRC, its expression ranging from 72% to 82% in the most recent published series [1Go–3Go]. Moreover, EGFR expression appears to be associated with poor survival and increased risk of invasion/metastasis [4Go] despite contradictory results [3Go]. These observations have all suggested EGFR as a rational target for molecular therapeutic strategies [5Go]. So, with the advent of these EGFR targeted therapies, particularly with monoclonal antibody-based therapies such as cetuximab that are directed to the cell surface domains of the receptor, clinical trials have increasingly used immunohistochemical screening strategies for EGFR expression to select patients with the highest likelihood of response to therapy. However, an important observation in the clinical studies so far conducted with cetuximab is the lack of correlation between expression of EGFR, as determined by immunohistochemistry (IHC), and clinical benefit, with objective responses also reported in EGFR-negative advanced CRC [6Go, 7Go]. One reason recently advocated to explain this lack of correlation was a possible difference in the EGFR status between the primary tumor and the metastatic sites [8Go].

There are few data in the literature regarding the EGFR status in the primary colon neoplasm and the corresponding distant metastases [8Go, 9Go]. The aim of our study was to analyze the correlation of immunohistochemical EGFR reactivity in the primary tumor and the related metastases.


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
Between 1995 and 2004, tumor specimens from 80 consecutive patients with CRC, who underwent surgery of the primary tumor and the corresponding metastatic site, were studied at the University Hospital Centre of Nice (France). Eighty-three consecutive CRC patients were initially identified through a pathological database. Three patients were excluded from analysis for the following reasons: tumor specimens not available (n=2) and carcinoid tumor (n=1).

EGFR IHC
All slides from the colon resection specimens and metastasectomies were reviewed by an experienced gastrointestinal pathologist. Immunostaining for EGFR was performed using the immunohistochemical system kit EGFR pharmDx (DakoCytomation, Carpinteria, CA, USA), on freshly cut, formalin-fixed, paraffin-embedded tissue. Paraffin-embedded tissues were cut at 5 µm thickness. Slides were dried at room temperature for 4 h and then placed at 57°C overnight. Sections were deparaffinized in two sequential xylene baths (5 min), 100% ethanol (3 min) and 95% ethanol (3 min), followed by a 5-min single wash in Wash-Buffer solution (Dako). The slides were loaded onto an autostainer (Dako) and the following washes and incubations were applied sequentially: 5-min buffer rinse, 5-min proteinase K incubation (Dako), 5-min buffer rinse, 5-min peroxydase blocking agent incubation, 5-min buffer rinse, 30-min primary EGFR antibody or negative control reagent incubation, 5-min buffer rinse, 30-min visualization reagent incubation, two buffer rinses, 5-min substrate chromogen solution incubation, and a final buffer rinse. Slides were removed from the autostainer and counterstained with hematoxylin for 10 min, then rinsed gently in reagent quality water. The positive and negative controls supplied by the manufacturer included pelleted, formalin-fixed, paraffin-embedded cell lines expressing grade staining of EGFR (line HT-29) and a negative cell line (CAMA-1).

EGFR status evaluation
Sections were analyzed using light microscopy by two observers who were unaware of the clinicopathological details, with simultaneous dual evaluation. Positivity for EGFR expression was defined as any membrane staining above background level. Both the primary and metastatic neoplasms were considered positive when ≥1% of the tumor cells had membranous staining. A specific membrane staining in <1% of neoplastic cells was defined as EGFR-negative. Cytoplasmic staining without associated membrane staining was reported as negative.

The intensity of EGFR reactivity in the adenocarcinoma cells was scored in the following manner (Figure 1): 1+ (weak intensity: faint brown membranous staining); 2+ (moderate intensity: brown membranous staining of intermediate darkness producing a complete or incomplete circular outline of the neoplastic cell); and 3+ (strong intensity: dark brown or black membranous staining producing a thick outline, complete or incomplete of the neoplastic cell). The level of EGFR expression was assessed according to the percentage of cells stained with weak or greater intensity as follows: 1–20%, 20–50% and >50%.



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Figure 1. Epidermal growth factor receptor intensity staining in primary colorectal tumors: (A) no membranous staining; (B) weak intensity (1+); (C) moderate intensity (2+); (D) strong intensity (3+). See text for details of staining intensity scoring.

 
Statistical analysis
The ‘R 1.7.1' statistical software package (the R Foundation for Statistical Computing, Vienna, Austria) was used for all calculations. Data management was performed using Microsoft Windows Access 2000 software. Pearson's correlation test confirmed by Spearman's correlation test was used to compare the EGFR staining between primary tumors and related metastatic sites (perfect correlation =1.0). Differences were considered to be statistically significant when the P value was ≤0.05. All statistical tests were two-sided.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical and pathological features
Eighty colorectal cancer patients comprised the study population. Patients’ characteristics are described in Table 1. The median age of the patients at initial surgery was 65 years (range 42–85). All patients had metastasis in one site. Eighty metastatic samples obtained from metastasectomies were analyzed, including 79 liver metastases and one pulmonary metastasis. Metastases were synchronous in 44 cases (55%) and metachronous in 36 cases (45%). The median time elapsed between resection of the primary and the corresponding metastatic site was 19 months. None of the patients had received prior EGFR-targeted therapy.


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Table 1. Patients’ characteristics (n=80)

 
EGFR reactivity of colorectal adenocarcinoma
Overall, 64 primary colorectal adenocarcinomas (80%) demonstrated EGFR reactivity in >1% of the adenocarcinoma cells (Table 2): 40 (50%) had EGFR expression in 1–20% of the adenocarcinoma cells, 13 (16.2%) had EGFR expression in 20–50% of the adenocarcinoma cells and 11 (13.8%) had EGFR expression in >50% of the adenocarcinoma cells. Twenty-four (30%) adenocarcinomas had 3+ intensity reactive cells, including 18 in <20% of the cells. EGFR reactivity was not uniform, with a greater percentage of neoplastic cells stained with a higher intensity at the invasion front of the adenocarcinoma. Sixteen patients (20%) had primary tumors that did not express EGFR.


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Table 2. Global results for epidermal growth factor receptor reactivity

 
EGFR reactivity in metastases
Sixty-five metastatic samples (81.3%) demonstrated reactivity in >1% of the metastatic cells (Table 2): 37 (46.2%) had expression in 1–20% of the cells, 14 (17.5%) had EGFR expression in 20–50% of the cells and 14 (17.5%) had EGFR expression in >50% of the cells. Twenty-three samples had 3+ intensity reactive cells, including nine in <20% of the tumor cells.

Among paired primary/metastatic tumors, only five (6.3%) showed discordance (Table 3): in two cases, EGFR was expressed in the primary tumor but not in the metastasis, and three samples showed EGFR expression in the metastasis but not in the primary tumor (Pearson correlation coefficient 0.8; 95% confidence interval 0.70–0.87; P<0.0001) (Figure 2).


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Table 3. Epidermal growth factor receptor status correlation between primary tumor and corresponding metastatic site

 


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Figure 2. (A) Primary colorectal tumor showing membranous epidermal growth factor receptor (EGFR)-positive staining. (B) Corresponding liver metastasis showing membranous EGFR-positive staining.

 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
CRC is the second leading cause of cancer death in the Western world. The last 10 years have seen a rapid evolution in metastatic CRC treatment, with a switch from 5-fluorouracil modulated by leucovorin to polychemotherapy including newer cytotoxic agents such as irinotecan and oxaliplatin. These regimens have increased time to progression and overall survival in patients with metastatic cancer [10Go, 11Go], and encouraging data are already available in the adjuvant setting [12Go]. Even with the significant improvement in traditional chemotherapy, there remain limitations with this treatment. Therefore, the last few years have witnessed growing interest and significant advances in the use of targeted therapy for CRC. Some of the most promising targets include the EGFR. Among the several potential strategies for targeting the EGFR, monoclonal antibodies and the low-molecular weight tyrosine kinase inhibitors are in the most advanced stages of clinical development. However, the level of EGFR expression required in the tumor to obtain clinical benefit from these therapies remains unknown at the present time. Moreover, the data with cetuximab in CRC showed that response rates were comparable in patients expressing 1+, 2+ or 3+ levels of EGFR [1Go, 2Go]. One mechanism advocated recently to explain the apparent lack of correlation between EGFR status and efficacy of EGFR-targeted therapy with monoclonal antibodies was the possible difference in EGFR status between the primary tumor (which was usually tested in clinical studies) and the metastatic sites, thus making cancer cells in these sites resistant to therapy [8Go]. Our results do not support this hypothesis. Indeed, between the paired primary tumor and distant metastatic lesions, 93.8% of samples had concordant EGFR status.

Nevertheless, our results are in disagreement with the recent study which showed that EGFR status did not correlate with EGFR expression in related metastatic sites [8Go]. In both studies, immunohistochemical analysis was performed using the same technical and interpretation procedures. However, in the study reported by Scartozzi et al. [8Go], 36% of primary tumors expressing EGFR showed a loss of expression in the corresponding metastatic site versus only 3% in our study. Our results seem to agree with preclinical data suggesting that EGFR expression is required for the tumor to acquire metastatic potential [13Go–15Go]. Nevertheless, in another study, McKay et al. [9Go] showed that EGFR expression was not identical in colorectal tumors and related lymph node metastases.

Other reasons could be advocated to explain why no clear association has emerged between the level of EGFR and the response to cetuximab in CRCs. Some of them are related to the ability to detect the target molecule in the tumor sample. EGFR expression can be evaluated in different ways by quantitation of the receptor at the DNA, RNA or protein level. IHC is commonly used to evaluate EGFR protein levels and is arguably the most convenient method for analyzing clinical samples. However, this method is not strictly quantitative, as there is no uniform scoring system and the interpretation of staining intensity is highly subjective. In addition, variations in protocols, such as in fixation procedures and antibodies, are likely to affect the sensitivity of these assays, making comparison of results from different laboratories difficult [16Go]. EGFR staining intensity declines dramatically with increasing storage time of the tissue samples, leading to false-negative samples. An alternative hypothesis to explain why IHC is a poor screening method for identifying patients with CRC who will respond to cetuximab therapy lies in the biology of EGFR. It is plausible, for example, that specific receptor sequences in antibody-binding extracellular domains will be identified that impact response to cetuximab. Finally, the lack of correlation between EGFR status and response to therapy could also be explained by the possibility that, in some cancer cells, regardless of the level of EGFR expression, the critical downstream signals may be activated via other receptors, or by other pathways [17Go–19Go]. So, a significant current challenge in EGFR-targeted therapy is to better identify those tumors in which growth is ‘dominantly driven’ by EGFR signaling.

Even if the current method to select CRC patients most likely to benefit from EGFR inhibition is probably inadequate, this unsuitability seems to be independent of the type of neoplastic tissue (primary tumor or metastasis) used for IHC assessment of the EGFR status. Establishing methods to identify tumors that truly depend on EGFR signaling for growth may represent a key to future progress.


    Acknowledgements
 
We wish to thank Dr Gerard Milano and Dr Emmanuel Chamorey for their helpful discussions.

Received for publication April 1, 2005. Revision received May 10, 2005. Accepted for publication May 12, 2005.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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2. Saltz LB, Meropol NJ, Loehrer PJ et al. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol 2004; 22: 1201–1208.[Abstract/Free Full Text]

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4. Hemming AW, Davis NL, Kluftinger A et al. Prognostic markers of colorectal cancer: an evaluation of DNA content, epidermal growth factor receptor, and Ki-67. J Surg Oncol 1992; 51: 147–152.[ISI][Medline]

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6. Lenz HJ, Mayer RJ, Gold PJ et al. Activity of cetuximab in patients with colorectal cancer refractory to both irinotecan and oxaliplatin. Proc Am Soc Clin Oncol 2004; 23 (248) (Abstr 3510).

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8. Scartozzi M, Bearzi I, Berardi R et al. Epidermal growth factor receptor (EGFR) status in primary colorectal tumors does not correlate with EGFR expression in related metastatic sites: implications for treatment with EGFR-targeted monoclonal antibodies. J Clin Oncol 2004; 22: 4772–4778.[Abstract/Free Full Text]

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16. Atkins D, Reiffen KA, Tegtmeier CL et al. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections. J Histochem Cytochem 2004; 52: 893–901.[Abstract/Free Full Text]

17. Prenzel N, Zwick E, Daub H et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999; 402: 884–888.[CrossRef][ISI][Medline]

18. Liu D, Ghiso JA, Estrada Y et al. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 2002; 1: 445–457.[CrossRef][ISI][Medline]

19. Carpenter G. Employment of the epidermal growth factor receptor-independent signaling pathways. J Cell Biol 1999; 146: 697–702.[CrossRef][ISI][Medline]





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