Annals of Oncology Advance Access originally published online on June 9, 2006
Annals of Oncology 2006 17(12):1850-1851; doi:10.1093/annonc/mdl138
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© 2006 European Society for Medical Oncology
letters to the editor |
EGFR expression in colon cancer: a break in the clouds
1 Centre Antoine-Lacassagne, Nice, France
2 Centre Jean-Perrin, Clermont Ferrand, Nice, France
3 Hôpital l'Archet, Nice, France
* (E-mail: gerard.milano{at}nice.fnclcc.fr)
Advances in the understanding of tumor biology have led to the development of targeted therapies [1]. One of the most promising is the epidermal growth factor receptor (EGFR), a member of the subclass I of the transmembrane receptor tyrosine kinase super-family [2] which is overexpressed in different solid cancers, reaching up to 80% overexpression in colorectal cancer [3, 4]. We recently analyzed EGFR in 82 colorectal cancer samples, based on EGF binding, and showed that in the majority of tumors EGFRs correspond to a single class of high-affinity binding sites, whereas approximately 20% of tumors exhibit additional low-affinity binding sites [5]. Experimental studies have shown that high-affinity EGFRs are the predominant biologically active receptors leading to tyrosine kinase activation whereas low-affinity EGFRs do not contribute significantly to intracellular signal activation [6].
A major clinical impact of EGFR targeting is illustrated by the results of the irinotecan-cetuximab trials showing that cetuximab alone, or combined with irinotecan, improved responsiveness in patients with irinotecan-refractory colorectal cancer [7, 8]. Both trials failed to establish a significant link between the intensity of EGFR tumor expression based on immunohistochemistry (IHC) and response to treatment. In a small retrospective study, Chung et al. provocatively reported a 25% objective response rate in EGFR-IHC negative colon cancer patients receiving cetuximab-based therapy [9]. Since the anti-EGFR antibodies currently used in IHC do not distinguish between high- and low-affinity EGFR [9], the authors hypothesized that tumors with few overall EGFRs, below the threshold of IHC detection but with a high ratio of high-affinity to low-affinity EGFR, may be responsive to anti-EGFR therapies.
The above considerations led us to extend our previous study [5], by comparing EGFR tumoral expression analyzed by means of both ligand binding assay (Scatchard analysis) and IHC method. This was done on a retrospective series of 28 colon cancer samples (11 women, 17 men, mean age: 68 years) corresponding to various tumor stages (two stages 0, two stages I, 10 stages II, 12 stages III and two stages IV). This study obtained approval from the local ethics committee and patients gave consent for tissue collection and analyses. Tumor samples (100 to 200 mg) were frozen and stored in liquid nitrogen. All analyzed samples were systematically controlled by histopathology. IHC was performed using Ventana 3C6 EGFR antibody following the manufacturer's recommendations. This antibody was selected for the present study because, in our hands, it gave the highest level of positive EGFR immunostainings in colorectal cancers when compared with the FDA approved Dako, pharmaDx kit [10]. Scatchard analyses were performed according to our previously published method, using human recombinant EGF as the ligand [11].
High-affinity specific EGF binding-sites were present in all tumors (Figure 1). Twenty-two tumors out of the 28 (78.6%) exhibited a single class of high-affinity binding sites, with a median Kd value at 0.53 nM (range 0.15–1.80) and a median site number at 86 fmol/mg prot (range 11–310). Six samples (21.4 %) exhibited two classes of binding sites with median high-affinity Kd and site number at 0.25 nM and 20 fmol/mg prot, respectively, and median low-affinity Kd and site number at 11 nM and 46 fmol/mg prot, respectively. Figure 1 illustrates the relationship between the IHC score and the number of high-affinity sites. The greater the high-affinity binding site, the higher the IHC score (Spearman rank correlation: r = 0.518, P = 0.005, n = 28). However, in the two tumors with IHC score 0, significant concentrations of high-affinity EGFRs were observed (33 and 62 fmol/mg prot, with Kd at 0.45 and 0.84 nM, respectively). Also surprising is the observation that distribution of high-affinity site numbers was not significantly different between IHC score 1, 2 and 3 (Kruskal-Wallis comparison: p = 0.67). On the other hand, the three tumors expressing the highest high-affinity site number (310, 244 and 171 fmol/mg prot, respectively) were correctly classified in IHC score IV. Also interesting is the significant correlation observed between IHC scoring and the sum of high- plus low-affinity binding sites (r = 0.532, p = 0.004, unshown data), with a correlation coefficient even slightly improved as compared to the previous correlation considering high-affinity sites only. This latter observation suggests that the 3C6 EGFR clone used recognizes both high- and low-affinity sites.
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correspond to tumors with one class of binding sites and 
correspond to tumors with two classes of binding sites.In total, EGFR IHC analysis only partially reflects the presence of functional EGFRs. This may explain the lack of predictivity of IHC assay for EGFR targeted therapy in colorectal cancer, with cases of responsiveness reported in patients with low EGFR tumoral expression by IHC [9]. The present observation does not preclude the value of IHC as a predictive tool but sheds light on the importance of the validation of this method, the choice of the antibody and the predictive cut-off to be used.
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