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Journal of Clinical Oncology, Vol 22, No 23 (December 1), 2004: pp. 4772-4778 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.00.117 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 AntibodiesFrom the Clinica di Oncologia Medica, Istituto di Anatomia Patologica; Azienda Ospedaliera Ospedali Riuniti; Università Politecnica delle Marche, Ancona, Italy Address reprint requests to Stefano Cascinu, MD, Clinica di Oncologia Medica, Azienda Ospedaliera Ospedali Riuniti-Università Politecnica delle Marche, via Conca, 60,020 Ancona, Italy; e-mail: cascinu{at}yahoo.com
PURPOSE: We hypothesized that the detection of epidermal growth factor receptor (EGFR) expression performed in primary tumors for treatment with EGFR-targeted monoclonal antibodies could not always correlate with EGFR status in metastatic sites, thus making cancer cells in these sites resistant to therapy. The aim of our study was to correlate EGFR expression on primary tumors and related metastases in order to find out whether assessing EGFR status on primary cancer is to be considered an effective tool for planning treatment with EGFR-targeted antibodies.
PATIENTS AND METHODS: We retrospectively evaluated EGFR immunohistochemistry from primary tumors and related metastatic sites in 99 colorectal cancer patients. The site of primary tumor was colon in 77 patients (78%) and rectum in 22 patients (22%). Metastatic sites analyzed were liver in 84 patients (81%), lung in 13 patients (13%), bone in one patient (1%), and brain in five patients (5%). EGFR status was defined as positive if the percentage of malignant cells stained was RESULTS: EGFR status was positive in 53 primary tumors (53%). In 19 primary tumors expressing EGFR (36%), the corresponding metastatic site was found negative, whereas it was found positive in seven metastases (15%) from EGFR-negative primary cancers. The difference between these two groups of patients (ie, EGFR-positive to EGFR-negative v EGFR-negative to EGFR-positive) was statistically significant (P = .036). CONCLUSION: Our results suggest that the detection of the EGFR in primary colorectal cancer could be inadequate for planning therapy with EGFR-targeted monoclonal antibodies in a considerable proportion of both EGFR-positive and -negative primary tumors (36% and 15%, respectively).
The epidermal growth factor receptor (EGFR) is a 170,000-kDa trans-membrane glycoprotein involved in signaling pathways affecting cellular growth, differentiation, and proliferation.1 An abnormal expression of the EGFR has been described in many human tumors and implicated in the development and prognosis of malignancies, thus representing not only a possible prognostic markers but primarily a rational molecular target for a new class of anticancer agents.2-8 In particular, initial reports with the use of monoclonal antibodies directed against the ligand-binding site of the EGFR has shown promising results in the treatment of colorectal cancer and will hopefully expand in the future treatment options available to patients diagnosed with this deadly disease.9-11 In fact, an interesting level of activity for cetuximab, an antibody directed against the EGFR, has been reported by Saltz et al in patients with chemotherapy-refractory colorectal cancer, and has been more recently confirmed by the BOND study (Bowel Oncology with cetuximab antibody), which also suggested a higher efficacy when this targeted therapy is used in combination with chemotherapy.9,11 Few preclinical data hypothesized a linear correlation between the expression of EGFR in cancer cells and response to treatment with EGFR-targeted therapies.12 Nevertheless, this concept rapidly became a preconceived assumption; therefore, EGFR status in tumors has been assumed to represent either the rationale or the predictive factor of choice for the efficacy of this treatment approach. Immunohistochemical evaluation of EGFR expression is the method that has been most widely used for patients' inclusion in clinical trials investigating the use of EGFR-targeted antibodies. In most of the anti EGFR clinical trials reported so far, however, any level of EGFR immunohistochemical expression in tumor cells has been considered adequate for predicting sensitivity to therapy, irrespective of both the percentage of neoplastic cells stained and the intensity of the staining.9,11 Furthermore, in these trials, a considerable proportion of colorectal tumors with high EGFR expression has been found to be refractory to treatment, whereas patients with tumors showing low expression of EGFR were responsive to therapy. Surprisingly, objective responses have been reported even in EGFR-negative advanced colorectal tumors treated with monoclonal antibodies against EGFR.13 Taken together, these findings failed to demonstrate that the level of EGFR staining could have any role in predicting the response rate. Although several mechanisms could be advocated to explain the apparent lack of correlation between EGFR status and efficacy of EGFR-targeted therapy with monoclonal antibodies, it is also possible that the detection of EGFR expression, which is usually performed in the primary tumor, does not always correlate with the EGFR status in metastatic sites, thus making cancer cells in these sites resistant to therapy. The aim of our study was to verify potential changes in EGFR biology between primary- and metastatic-stage disease, by correlating EGFR immunohistochemical expression in primary colorectal tumors and corresponding metastases. This was done to find out whether assessing EGFR status in primary cancer cells could be considered an effective tool for planning therapy with EGFR-targeted monoclonal antibodies.
Patient Selection Patients were selected from a pathological database of colorectal cancer cases undergoing surgical resection of the primary tumor and the corresponding metastatic site and observed at the Pathology Department of the Università Politecnica delle Marche (Ancona, Italy) between 1995 and 2004.
Immunohistochemical Analysis Immunohistochemical staining of sections was performed using the immunohistochemical system kit EGFR pharmDx (DakoCytomation, Carpinteria, CA), which included proteinase K, peroxidase block, EGFR pharmDx monoclonal mouse antibody (clone 2-18C9), mouse IgG1 negative control reagent, labeled polymer HRP, DAB + substrate buffer, liquid DAB + chromogen, DakoCytomation wash solution 10x, and EGFR pharmDx control slides. The immunostaining and reagents storage was performed according to instructions supplied by the manufacturer. Reagent and control slides were stored at 2°C to 8°C and were equilibrated to room temperature (20°C to 25°C) before immunostaining. The sections were deparafinized and rehydrated with sequential wash in xylene, alcohol, and distilled water, and then loaded onto an autostainer (DakoCytomation). Washes and incubations were applied sequentially as described in Table 1. Subsequently, the slides were counterstained with Meyer's hematoxylin for 1 minute, and coverslipped. The controls used for the validation of EGFR assay were included in EGFR pharmDx: negative control reagent, positive and negative control cell preparation.
The negative control reagent is a monoclonal mouse antibody to an enzyme that is not expressed in mammalian tissues.18 The cell controls consist of sections of two pelleted formalin-fixed paraffin-embedded human cell lines. The cell line CAMA-1 (breast carcinoma) is devoid of EGFR expression; the cell line HT29 (colon adenocarcinoma) expresses the EGFR with moderate membrane staining (2+).18
Evaluation of EGFR Expression Cytoplasmic staining resulting from either internalized or nascent receptor molecules, without associated membrane staining, was reported as negative.18 The intensity of EGFR reactivity was scored using a three-tier system as follows19: 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; Fig 1).
Statistical Analysis The SAS (SAS Institute, Cary, NC) system was used for statistical analysis. Observed differences were considered significant if P < .05. Patients were examined to determine whether a statistically significant difference existed regarding EGFR status variation between paired primary tumor and metastatic site (positive to negative v negative to positive) and EGFR status variation, from EGFR-positive primary tumor to EGFR-negative metastasis, according to metastatic site (liver metastases v lung metastases;  2 test).
Ninety-nine patients were available for our analysis. There were 61 men (62%) and 38 women (38%); median age at diagnosis was 63 years (range, 32 to 86 years). Seventy-seven patients (78%) had colon cancer and 22 (22%) had rectal cancer. Colorectal adenocarcinoma was the most common histological type, as it was observed in 93 pathological samples (94%) from primary tumors; the remaining six cases (6%) were mucinous tumors. Histologic grade 1-2 and grade 3 were described in 84 (85%) and 15 tumors (15%), respectively. Ninety-five cases of primary neoplasms had metastasis in a single site, three cases had both pulmonary and hepatic metastasis, and only one case had hepatic and cerebral metastasis. Globally pathologic samples from 103 metastatic sites were analyzed, and included 84 liver metastases (81%), 13 lung metastases (13%), five brain metastases (5%), and one bone metastasis (1%; Table 2).
All metastatic samples were obtained from metastasectomies, except for the only bone metastasis, which was a biopsy. Liver metastases were synchronous in 43 cases and metachronous in the remaining 41 cases. Seven lung metastases were synchronous, whereas the remaining six were metachronous as they were all brain lesions (five cases). The median time elapsed between resection of the primary and corresponding metastatic site was 11 months. EGFR immunohistochemical expression status was positive in 53 primary tumors (53%); furthermore, in most of the EGFR-positive primary carcinomas (45 cases; 85%), the percentage of positive cancer cells was higher than 10%. Analysis of metastatic sites showed EGFR expression in 39 liver metastases (46%), in six lung metastases (46%), and in two brain lesions (40%; Table 3). The only bone metastasis available for analysis was EGFR-negative, as it was the corresponding primary tumor. The correlation analysis between primary cancer and corresponding metastatic sites revealed that in 19 EGFR-positive tumors (36%), the related metastatic site was EGFR-negative (Fig 2). On the contrary, in only seven EGFR-negative tumors (15%), cancer cells regained EGFR expression in metastases. The difference between these two groups of patients (ie, EGFR-positive to -negative v EGFR-negative to -positive) was statistically significant (P = .036; Table 4; Fig 3).
Advanced colorectal cancer continues to represent a major health problem worldwide, but the introduction of a novel class of targeted antineoplastic agents such as those directed against the EGFR has notably expanded the available therapeutic options for these patients.9-11 Few preclinical data suggested a linear correlation between the expression of EGFR in cancer cells and response to treatment with EGFR-targeted therapies.12 Nevertheless, this concept rapidly became a preconceived assumption, and therefore, EGFR status in tumors has been assumed to represent either the rationale or the predictive factor of choice for the efficacy of this treatment approach. Unfortunately, clinical data presented thus far have failed to demonstrate a correlation between the level of EGFR staining and the global outcome of patients treated, making the need for a reliable predictive factor in the pretreatment selection of potential candidates for EGFR-targeted monoclonal antibody therapy even more relevant.9,11,13 We retrospectively evaluated EGFR status on primary colorectal tumors and corresponding metastatic sites in order to assess whether the apparent lack of correlation between EGFR status and efficacy of EGFR-targeted therapy with monoclonal antibodies could be related, at least in some cases, to the loss of EGFR expression in metastatic cells. This biological phenomenon, if confirmed, could in part explain tumor resistance to EGFR targeted antibody therapy. Ninety-nine cases were available for our analysis. EGFR immunohistochemical expression in  1% of neoplastic cells was detected in 53 primary colorectal tumors (53%), with 45 cases (85%) showing a percentage of positive cells higher than 10%. These data are comparable to what has been previously reported in the published literature, which also, however, indicates a great variability in the percentage of EGFR-positive tumors, probably according to the method used and cutoff values chosen for defining EGFR expression.6-8,14,15 The correlation analysis between primary cancer and corresponding metastatic sites revealed that in 19 EGFR-positive tumors (36%), the related metastatic site was EGFR-negative. On the contrary, in seven EGFR-negative tumors (15%), cancer cells regained EGFR expression in metastases (Table 4). Other molecular mechanisms, such as activating EGFR mutations, increased ligand expression, alteration of downstream signaling pathways, heterodimerization, and cross-talk among different erB receptor family members, and can be advocated to explain resistance to EGFR-targeted therapies, but most of them are still under investigation, and available data are preliminary.5,16,17 The VEGF gene has been indicated as one of the possible downstream targets of EGFR kinase; thus, one of the putative antineoplastic effects derived from the inhibition of EGFR is thought to be the consequent downregulation of the angiogenic process, which is essential for tumor progression. Therefore, it has been postulated that the activation of pathways driving VEGF expression independently of EGFR might result in resistance to EGFR inhibitors.18,19 Although interesting, especially for its possible clinical application, this mechanism deserves further investigation before we can use it in planning targeted therapy in advanced colorectal cancer. The method used to determine EGFR expression could be crucial for the process aiming to establish a correlation between response to anti-EGFR targeted therapy and the presence/overexpression of the molecular target on cancer cells. Immunohistochemistry evaluation, according to a previously described standard procedure, represented the technique that has been most widely used in clinical trials investigating the medical use of monoclonal antibodies against EGFR. Nevertheless, the role of staining characteristics such as the pattern of cellular membrane staining, complete or incomplete, that are considered relevant in other neoplastic conditions involving the use of targeted antibodies (ie, c-erb B2 expression in breast cancer for trastuzumab therapy), has never been reported. The finding that EGFR status may change between primary tumor and corresponding distant metastatic sites is novel and brings new insights into the biology of EGFR expressing colorectal tumors. This implies that, at least hypothetically, this biologic phenomenon could account for resistance to monoclonal antibody directed against the EGFR in a non-negligible proportion of cases if we assume that the loss of the target (EGFR) should render ineffective any treatment directed against it. McKay et al have previously reported that EGFR expression was not identical in colorectal tumors and related lymph node metastases.20 However, thus far, no data are available on EGFR levels in paired primary cancer and distant metastatic sites, which could be, on the contrary, highly relevant for treatment implications. Loss of EGFR expression could be a result of genetic abnormalities occurring in tumors, such as chromosomal deletions or gene silencing. Although silencing of the EGFR gene has not been reported to date, loss of amplified EGFR due to chromosomal deletion has been observed in EGFR-overexpressing cells, in the presence of a high concentration of epidermal growth factor.16,21 Since EGFR-targeted monoclonal antibodies are used to treat metastatic disease, if EGFR status is to be used for selection or exclusion from therapy, on the basis of our data, only EGFR status in metastases would be relevant. Nevertheless, only a prospective trial including EGFR assessment on metastases could definitely establish whether this could be considered appropriate in clinical practice. After initial reports of activity with EGFR-targeted monoclonal antibodies in advanced colorectal cancer, the debate about the ideal definition for the pretreatment prediction of colorectal cancer sensibility to therapy has just begun. Currently, treatment decisions in this area are usually being made on the basis of EGFR immunohistochemistry results on primary colorectal tumor. This implies that, in clinical practice, some patients are getting this treatment option because of a detectable EGFR expression, and others are being denied the same option because of an absent EGFR expression. However, our findings showing a substantial EGFR status difference between primary tumor and related metastatic sites, suggest that the current practice of immunohistochemical analysis of any available neoplastic tissue should probably be considered inadequate in a non-negligible proportion of cases.
The authors indicated no potential conflicts of interest.
Preliminary results of this analysis have been submitted to the European Society of Medical Oncology for presentation at the 2004 meeting. Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. Carpenter G, Cohen S: Epidermal growth factor. J Biol Chem 265:7709-7712, 1990 2. Sato JD, Kawamoto T, Le AD, et al: Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med 1:511-529, 1983[Medline] 3. Goldstein NI, Prewett M, Zuklys K, et al: Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res 1:1311-1318, 1995[Abstract] 4. Laskin JJ, Sandler AB: Epidermal growth factor receptor: A promising target in solid tumours. Cancer Treat Rev 30:1-17, 2004[CrossRef][Medline] 5. Ciardiello F, Tortora G: Epidermal growth factor receptor (EGFR) as a target in cancer therapy: Understanding the role of receptor expression and other molecular determinants that could influence the response to anti-EGFR drugs. Eur J Cancer 39:1348-1354, 2003 6. Kluftinger AM, Robinson BW, Quenville NF, et al: Correlation of epidermal growth factor receptor and c-erbB2 oncogene product to known prognostic indicators of colorectal cancer. Surg Oncol 1:97-105, 1992[CrossRef][Medline] 7. 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 51:147-152, 1992[Medline] 8. Mayer A, Takimoto M, Fritz E, et al: The prognostic significance of proliferating cell nuclear antigen, epidermal growth factor receptor, and mdr gene expression in colorectal cancer. Cancer 71:2454-2460, 1993[CrossRef][Medline]
9. Saltz LB, Meropol NJ, Loehrer PJ Sr, et al: Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol 22:1201-1208, 2004
10. Ellis LM, Hoff PM: Targeting the epidermal growth factor receptor: An important incremental step in the battle against colorectal cancer. J Clin Oncol 22:1177-1179, 2004
11. Cunningham D, Humblet Y, Siena S, et al: Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 351:337-345, 2004
12. Castillo L, Etienne-Grimaldi MC, Fischel JL, et al: Pharmacological background of EGFR targeting. Ann Oncol 15:1007-1012, 2004 13. 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 23:248, 2004 (abstr 3510) 14. Spaulding DC, Spaulding BO: Epidermal growth factor receptor expression and measurement in solid tumors. Semin Oncol 29:45-54, 2002 15. Goldstein NS, Armin M: Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: Implications for a standardized scoring system. Cancer 92:1331-1346, 2001[CrossRef][Medline] 16. Viloria-Petit AM, Kerbel RS: Acquired resistance to EGFR inhibitors: Mechanisms and prevention strategies. Int J Radiat Oncol Biol Phys 58:914-926, 2004[CrossRef][Medline]
17. Christensen JG, Schreck RE, Chan E, et al: High levels of HER-2 expression alter the ability of epidermal growth factor receptor (EGFR) family tyrosine kinase inhibitors to inhibit EGFR phosphorylation in vivo. Clin Cancer Res 7:4230-4238, 2001
18. Ciardiello F, Bianco R, Damiano V, et al: Antiangiogenic and antitumor activity of anti-epidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial growth factor antisense oligonucleotide in human GEO colon cancer cells. Clin Cancer Res 6:3739-3747, 2000
19. Viloria-Petit A, Crombet T, Jothy S, et al: Acquired resistance to the antitumor effect of epidermal growth factor receptor-blocking antibodies in vivo: A role for altered tumor angiogenesis. Cancer Res 61:5090-5101, 2001 20. McKay JA, Murray LJ, Curran S, et al: Evaluation of the epidermal growth factor receptor (EGFR) in colorectal tumours and lymph node metastases. Eur J Cancer 38:2258-2264, 2002
21. Filmus J, Trent JM, Pollak MN, et al: Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants. Mol Cell Biol 7:251-257, 1987 Submitted August 24, 2004; accepted September 1, 2004.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
