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
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Abstract |
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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
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Introduction |
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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 [13
]. Moreover, EGFR expression appears to be associated with poor survival and increased risk of invasion/metastasis [4
] despite contradictory results [3
]. These observations have all suggested EGFR as a rational target for molecular therapeutic strategies [5
]. 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 [6
, 7
]. 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 [8
].
There are few data in the literature regarding the EGFR status in the primary colon neoplasm and the corresponding distant metastases [8, 9
]. The aim of our study was to analyze the correlation of immunohistochemical EGFR reactivity in the primary tumor and the related metastases.
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Patients and methods |
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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: 120%, 2050% and >50%.
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Results |
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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 0.700.87; P<0.0001) (Figure 2).
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Discussion |
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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 [8]. In both studies, immunohistochemical analysis was performed using the same technical and interpretation procedures. However, in the study reported by Scartozzi et al. [8
], 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 [13
15
]. Nevertheless, in another study, McKay et al. [9
] 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 [16]. 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 [17
19
]. 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.
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Acknowledgements |
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Received for publication April 1, 2005. Revision received May 10, 2005. Accepted for publication May 12, 2005.
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References |
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