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The R521K polymorphism of epidermal growth factor receptor has attenuated affinity in ligand binding and proto-oncogene induction, which may affect the efficacy of cetuximab. We analyzed the effect of this polymorphism on the outcome of 112 patients with KRAS wild-type metastatic colorectal carcinoma treated with first-line cetuximab plus FOLFOX-4. The associations of this polymorphism with vascular endothelial growth factor (VEGF) expression and clinicopathologic characteristics were also examined. The results showed that the frequencies of the G/G, G/A, and A/A genotypes were 32.1% (n = 36), 42.9% (n = 48), and 25.0% (n = 28), respectively. A marked decrease in VEGF expression levels (66.7% vs 28.9%, P < 0.01) was observed in patients with 521A allele variants (Arg/Lys or Lys/Lys), which were associated with a decreased tumor size (55.6% vs 31.6%, P = 0.02), good histological differentiation (63.9% vs 85.5%, P = 0.01), decreased lymphovascular invasion (69.4% vs 39.5%, P < 0.01), and a higher response rate to cetuximab plus FOLFOX treatment (55.6% vs 78.9%, P = 0.01). In addition, this polymorphism was associated with a longer progression-free period (P = 0.001) and overall survival (P = 0.001). By multivariate analysis, this polymorphism was also identified as an independent prognostic factor. These data suggest that the R521K polymorphism of epidermal growth factor receptor, by reducing its activation and a consequential downregulation of its target genes, including VEGF, could be a key determinant of an increased response to cetuximab-based chemotherapy and a longer survival for KRAS wild-type colorectal carcinoma patients. (Cancer Sci 2012; 103: 791–796)
Cetuximab, a chimeric mAb, is an antibody against the extracellular domain of epidermal growth factor receptor (EGFR). It binds to EGFR with a high affinity and is able to compete with epidermal growth factor (EGF) binding, thereby inhibiting subsequent receptor activation and signalling. Cetuximab is approved for the treatment of patients with metastatic colorectal carcinoma (CRC) and squamous carcinoma of the head and neck. A favorable effect of cetuximab combined with chemotherapy in advanced non-small-cell lung cancer (NSCLC) has also been reported. The benefits of cetuximab-based therapies are restricted to a particular subgroup of patients. The EGFR expression, as evaluated by immunohistochemistry, does not correlate with the response to cetuximab, but an increase in EGFR copy number identified by FISH, may predict a better response. Loss of phosphatase and tensin homolog (PTEN) protein expression is also associated with a lower response rate.
Mutations of KRAS, a gene encoding a G protein that plays a key role in the downstream signaling of EGFR, lead to resistance to cetuximab. Mutations of KRAS occur in approximately 40% of CRC, and the mutation status of KRAS is considered a good predictive marker for cetuximab-based treatment. Mutations of the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and PIK3CA genes are also associated with a lower response rate to cetuximab, and the prediction of response rates could be improved by additional genotyping of these genes.
In KRAS wild-type populations, there remains a subgroup of patients who do not respond to cetuximab; therefore, additional markers for predicting the response are indicated. Genetic variations in EGFR or its ligand, EGF, may predict clinical outcome in patients treated with cetuximab-based regimens.[8, 9] For example, the A61G polymorphism of EGF predicts the effect of cetuximab plus irinotecan. In addition, the intron 1 CA dinucleotide repeat polymorphism of EGFR predicts survival of KRAS wild-type patients treated with cetuximab-based chemotherapy.
A polymorphic variant in EGFR arising from a single nucleotide substitution (142285 G>A), leading to an arginine (R)/lysine (K) substitution in codon 521 in the extracellular domain of EGFR, has been identified. This polymorphism, previously described as R497K according to an older nomenclature, was found to be associated with a lower pelvic recurrence in rectal cancer patients treated with chemoradiation, longer survival in stage II/III CRC patients who received curative surgery, and a better response to oxaliplatin-based chemotherapy. Compared with the wild-type 521R allele, the 521K allele variant has attenuated affinity in ligand binding, tyrosine kinase activation, and induction of the proto-oncogenes myc, fos, and jun. In breast cancer patients, the 521K allele variant was found to be associated with decreased lymph node metastasis and good histological differentiation. Interestingly, this polymorphism was associated with progression-free survival in CRC patients treated with single-agent cetuximab. In a retrospective study of 32 CRC patients with different KRAS status treated with cetuximab plus irinotecan, this polymorphism was found to be associated with higher response rate and longer survival; however, the enrolled patients were quite heterogeneous.
Based on these earlier findings, we propose that the R521K polymorphism of EGFR might be associated with a better outcome in CRC patients receiving cetuximab-based treatments. A study was carried out in 112 KRAS wild-type CRC patients treated with first-line cetuximab plus FOLFOX-4, and the potential of using this polymorphism as a predictive/prognostic marker was evaluated.
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Somatic mutations of EGFR are associated with increased sensitivity to EGFR-tyrosine kinase inhibitors in NSCLC, but such mutations are rare or absent in CRC, and the addition of gefitinib to chemotherapy does not improve therapeutic efficacy in CRC patients.KRAS mutations lead to resistance to cetuximab; but in KRAS wild-type populations, there remains a subgroup of patients that does not respond to cetuximab. Polymorphisms in EGFR have attracted a lot of attention because they affect not only their function in ligand binding, but also clinicopathologic features and response to current therapeutic agents,[11, 12] including cetuximab.[4, 15] In the current study, we focused on the extracellular domain of EGFR because this region has been shown to be highly polymorphic and to have a significant impact on the function in ligand binding and subsequent signaling. The R521K variant has also been described as being associated with cancer severity in EGFR-expressing tumors, such as gliomas and lung cancer.
In a retrospective study of 32 patients with metastatic CRC treated with cetuximab plus irinotecan, the R521K polymorphism of EGFR was found to be associated with higher response rate. However, the enrolled patients were quite heterogeneous. In 22 (68.8%) patients, cetuximab/irinotecan was given after two or more regimens failed, and a variety of previous chemotherapy was used in different individuals. One of them was even chemotherapy-naive. In addition, the enrolled patients had different KRAS statuses, including 14 (43.8%) patients with KRAS mutations and another 18 with wild-type KRAS, making difficult to ascertain the predictive/prognostic value of this EGFR polymorphism in this study.
The R521K (or R497K according to an older nomenclature) polymorphism of EGFR was previously found to be a prognostic marker for CRC patients because it was associated with longer survival in stage II/III CRC patients treated with curative surgery. In the current study, we further identified that this polymorphism could serve as a predictive marker for cetuximab-based treatment in KRAS wild-type CRC. We found that the EGFR R521K polymorphism correlates with a higher response rate (Table 2) and a favorable prognosis (Fig. 4). An attenuated EGFR signaling induced by the R521K polymorphism that could be more sensitive to targeted receptor inhibition was proposed. The resulting amino acid substitution, arginine to lysine, has been shown to reduce ligand binding and ligand-induced EGFR signaling. Therefore, this genetic variant may alter the binding of its specific ligands, leading to an altered phenotype of EGFR signaling. Of particular interest, the expression of EGFR ligands epiregulin and amphiregulin, which may predict cetuximab benefit, has also been clearly shown. In fact, the resulting amino acid substitution is located at the boundary between EGFR domain III, which represents the direct interaction site with cetuximab, and domain IV. Therefore, the 521K variant could affect cetuximab binding and/or effects.
In the current study, G/A or A/A genotypes in the CRC patients were associated with smaller tumor size, better histologic differentiation, and lower rate of lymphovascular invasion (Table 1). These data are compatible with a previous report that the R521K polymorphism is correlated with a lower tumor grade and fewer lymph node metastases, and might be a prognostic factor in breast cancer patients. The association between the EGFR R521K polymorphism and lymph node metastasis deserves further consideration as a clinical indicator during pre-operative evaluation. In fact, various studies have considered genetic factors involving cell mobility, vascular invasion and angiogenesis for predicting lymph node metastasis.
We found that the R521K polymorphism of EGFR correlates with a higher response rate to cetuximab-based treatment and a favorable prognosis in KRAS wild-type CRC patients (Table 2, Fig. 4). However, patients with this polymorphism also have lower VEGF expression levels (Fig. 3); therefore, the confounding bias between EGFR polymorphism and VEGF status should be considered. Epidermal growth factor receptor signaling regulates the synthesis of several pro-angiogenic growth factors, including VEGF. Previous studies have shown that the expression of VEGF is related to the extent of tumor vascularization and prognosis, and is predictive of resistance to chemotherapy. Interestingly, in a subset of human tumors, VEGF may promote the malignant progression of tumor cells by directly acting on its receptors through an endothelial cell-independent pathway.[30, 31] The expression of VEGF receptor 1 (VEGFR1) and VEGFR2 has been shown in CRC cells, and the activation of these receptors by VEGF leads to activation of the MAPK pathway and phenotypic changes in CRC cells.[30, 31] Clinically, the addition of bevacizumab, a mAb directed against VEGF, to chemotherapy improves response rates and survival for patients with metastatic CRC.
Discrepancies in the association between genotype of EGFR codon 521 and its impact on clinical outcome of anti-EGFR therapy exist in different types of malignancy. The R521K polymorphism was associated with a longer progression-free survival in CRC patients treated with cetuximab. In patients with NSCLC treated with gefitinib, although sensitivity to treatment strongly depends on the EGFR mutation status, the R521K polymorphism of EGFR was not associated with response rate. Whether this is due to different drugs used for treatment, or simply due to different tumor types, deserves further study. In addition, because FOLFOX-4 was used in combination with cetuximab in the current study, it remains possible that the improvement in survival seen in patients with G/A and A/A genotypes is not a reflection of increased sensitivity to cetuximab, but rather that this group inherently has a higher sensitivity to FOLFOX-4, or a longer survival owing to decreased EGFR signaling.
Compared with Caucasian populations, a remarkably higher prevalence (67.9%) of the EGFR 521K allele variants was noted in our patients. Ethnic differences have a profound influence on the response and toxicity to chemotherapy in malignant diseases. For example, the UGT1A1*28 polymorphism is rare in Asian populations, which leads to a decreased risk of developing severe neutropenia after being treated with irinotecan. Due to a higher prevalence of EGFR mutations, gefitinib is very effective in Asian patients with NSCLC. In the current study, the frequency of EGFR codon 521 A allele variants was 67.9%, including 42.9% G/A and 25.0% A/A genotypes, which was significantly higher than that in Caucasian (37.5%) and Tunisian (39.2%) populations.[4, 14] This result implied that Asian populations might have an attenuated EGFR ligand binding affinity and a better outcome to cetuximab-based treatment, which deserves further study.
The activation of EGFR initiates intracellular proliferation signaling results in proliferation and survival through the Ras/Raf/MEK/ERK or PI3K/PTEN/AKT pathways, respectively. Therefore, alterations of the downstream effectors of the EGFR pathway may have influence in its signaling and the efficacy of EGFR-targeted therapies. Loss of PTEN protein expression, and mutation of BRAF have been associated with lower efficacy of therapies directed against EGFR-activated pathways. However, mutations in BRAF were identified in less than 5% of CRC patients, and this mutation is not routinely examined in clinical practice. Mutation of the PIK3CA gene, resulting in a mutant PI3-kinase and constitutively activated Akt signaling, is also associated with a lower response rate to cetuximab. The prediction of response rates to cetuximab could be improved by additional genotyping of these genes, and prospective studies in clinical trial cohorts will be required to confirm the utility of these markers.
In summary, we showed that the R521K polymorphism of EGFR is associated with decreased VEGF expression in CRC cells. By reducing the activation of EGFR and consequential downregulation of its target genes, this polymorphism is likely to be one determinant of increased response to cetuximab-based chemotherapy and longer survival for KRAS wild-type CRC patients.