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Journal of Clinical Oncology, Vol 23, No 15 (May 20), 2005: pp. 3536-3544 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.09.100 Molecular Determinants of Cetuximab EfficacyFrom the Division of Medical Oncology, Department of Preventive Medicine, Department of Pathology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine; and Response Genetics Inc, Los Angeles, CA Address reprint requests to Heinz-Josef Lenz, MD, FACP, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, 1441 Eastlake Ave, Suite 3456, Los Angeles, CA 90033; e-mail: lenz{at}usc.edu
PURPOSE: To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS: There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION: This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.
Colorectal cancer (CRC) is the third most common malignant tumor in the United States. In 2004 an estimated 146,940 new cases and 56,730 deaths will occur.1 The median overall survival of patients with advanced CRC has increased from 12 months to about 18 to 21 months in the past decade.2 This improvement is mainly based on the introduction of two chemotherapeutic drugs in the treatment of CRC—oxaliplatin and irinotecan (CPT-11).3,4 Even with the significant improvement in traditional chemotherapy, there remain limitations with this treatment. Therefore, several novel targets are being investigated both as single agents and in combination with chemotherapy to assess the potential for increased efficacy. One of the most promising targets is the epidermal growth factor receptor (EGFR), a member of the type I receptor tyrosine kinase family. EGFR is over-expressed in different solid cancer types, including up to 77% of CRC and is involved in malignant transformation and tumor growth through the interference of apoptosis, cellular proliferation, angiogenesis, and metastasis.5 Overexpression of EGFR has been associated with poor prognosis and has been suggested as a potential target for antitumor agents.5-8 Studies have shown that monoclonal antibodies directed at the EGFR inhibit the growth of EGFR-expressing cancer cells.5 Monoclonal antibodies represent one of the most important options in the inhibition of EGFR. They specifically bind to the domain of the receptor and competitively inhibit ligand binding, inducing receptor dimerization and down-regulation.9,10 The most promising monoclonal antibody to date is cetuximab (Erbitux; ImClone Systems Inc, York, NY). There are multiple mechanisms that are thought to contribute to the antitumor activity of cetuximab. The addition of this monoclonal antibody to tumor cell cultures has been shown to result in a direct inhibition of receptor tyrosine kinase activity.11 Furthermore, it has been shown that the blockade of the EGFR signaling pathway lead to an activation of proapoptotic mechanisms, the induction of antimetastatic and anti-invasive properties, and to a significant inhibition of angiogenesis.11 Several clinical phase II studies have shown that cetuximab has promising effects in patients with advanced/metastatic CRC.2,12-14 In a European multicenter trial, Cunningham et al enrolled 329 patients with CRC that progressed under CPT-11-based therapy.2 Patients received either a combined therapy with CPT-11 and cetuximab or a monotherapy with the monoclonal antibody. The combined therapy with cetuximab/CPT-11 produced significantly greater efficacy than the single-agent therapy, with a higher objective response rate (22.9% v 10.8%, respectively), overall disease control rate (55.5% v 32.4%, respectively), and a prolonged time to progression (4.1 v 1.5 months, respectively). Saltz et al enrolled 57 patients with EGFR-expressing CRC in a phase II trial that progressed on CPT-11 treatment.12 All patients were treated with single-agent cetuximab. Of all patients, 9.5% had a partial response, and 35% had a minor response or stable disease. Until now, there have been no reliable markers to identify patients who will most likely benefit from this therapy. Both the trials of Cunningham and Saltz have failed to show a significant correlation between EGFR expression based on immunhistochemistry (IHC) and response to treatment with either cetuximab and CPT-11, or cetuximab alone.2,12 Interestingly, in this phase II multicenter trial, one of nine patients with undetectable levels of EGFR protein expression responded to cetuximab therapy.13 To date, only skin toxicity has been shown to be significantly associated with response and overall survival in patients with metastatic CRC receiving cetuximab treatment.2,12 Our goal was to obtain preliminary data on molecular predictors of cetuximab therapy. We tested the hypothesis that EGFR gene expression levels are associated with response, survival and toxicity independent of skin toxicity in patients with metastatic CRC treated with cetuximab. Furthermore, we analyzed the mRNA levels of enzymes involved in the EGFR signaling pathway: cyclo-oxygenase 2 (Cox-2), which is thought to be a regulator of EGFR, as well as cyclin D1 (CCND1), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), which are thought to be effectors of EGFR (Fig 1).15-18
Patient Selection and Chemotherapy Treatment All patients from the University of Southern California/Norris Comprehensive Cancer Center (Los Angeles, CA) who took part in the phase II open-label multicenter study (IMCL-0144) of cetuximab were included in our pilot study because these 39 patients signed an additional informed consent for tissue and blood collection to study molecular correlates.13 The patients included had histopathologically confirmed metastatic CRC, had failed at least two prior chemotherapy regimens or adjuvant therapy plus one chemotherapy regimen for metastatic disease, and had disease progression within 6 months of completing adjuvant therapy. The study was investigated at University of Souther California/Norris Comprehensive Cancer Center and was approved by the institutional review board of the University of Southern California for Medical Sciences. All patients had IHC evidence of EGFR expression in their tumor samples. Patients were treated with cetuximab at a standard loading dose of 400 mg/m2 over 2 hours, followed by weekly 250-mg/m2 treatment over 1 hour. Treatment was continued until progression of disease or toxicity occurred and patients were evaluated every 6 weeks for tumor response. For the evaluation of gene expression levels, tumor samples were obtained from the primary colorectal tumor or from metastatic site at the time of diagnosis.
Microdissection
RNA Isolation and cDNA Synthesis
Real Time Polymerase Chain Reaction Quantification of mRNA expression
TaqMan measurements yield cycle threshold (Ct) values that are inversely proportional to the amount of cDNA in the tube (ie, a higher Ct value means more PCR cycles are required to reach a certain level of detection). Gene expression values (relative mRNA levels) are expressed as ratios (differences between the Ct values) between the gene of interest and an internal reference gene (ß-actin) that provides a normalization factor for the amount of RNA isolated from a specimen.
IHC
Statistical Analysis
Gene expression values are expressed as ratios between two absolute measurements—the gene of interest and the internal reference gene, ß-actin. The associations between gene expression levels and response to cetuximab (partial response (PR), stable disease, and progressive disease) and toxicity (grade 0-1 v grade 2 to 3) were evaluated by nonparametric methods (the Kruskal-Wallis test for response and the Mann-Whitney U test for toxicity). To assess the associations between the expression level of each gene and overall survival, the expression level was categorized into a low and a high value at optimal cut points. The maximal All reported P values were two-sided. All analyses were performed using the SAS statistical package version 8.2 (version 8; SAS Institute Inc, Cary, NC) and Epilog Plus Version 1.0 (Epicenter Software, Pasadena, CA).
A total of 39 patients were enrolled on this study, including 21 women and 18 men, with a median age of 64 years (range, 35 to 83 years). Thirty-one patients (79%) were white, six (15%) were Asian, and two (5%) were Hispanic. All patients were assessable to associate EGFR expression detected by IHC staining to parameter of clinical outcome. Due to limited tissue sampling, 34 patients (87%) were assessable to associate gene expression levels of Cox-2, CCND1, EGFR, IL-8, and VEGF to response, survival, and toxicity. The median follow-up was 7.1 months (95% CI, 2.5 to 21.6 months), and the median survival time was 5.5 months (95% CI, 2.7 to 8.7 months). Two patients (6%) had PR, 21 patients (60%) had stable disease, and 12 patients (34%) had progressive disease under treatment with cetuximab, while no patient had complete response, and for four patients, the response was not assessable. Skin reactions were observed in 85% of the 39 patients, of whom 12 (31%) had grade 1, 20 (51%) had grade 2, and one (3%) had grade 3 skin reactions (Table 2).
Gene Expression Levels of Cox-2, CCND1, EGFR, IL-8, and VEGF Cox-2 gene expression was quantifiable in 27 (79%) of the 34 samples; CCND1 expression, in 31 samples (91%); EGFR expression, in 30 samples (88%); IL-8 expression, in 33 samples (97%); and VEGF expression, in 31 samples (91%). The reason for these different gene expression levels is due to the low or limited amount of cDNA/RNA generated from these microdissected paraffin-embedded tissues. The median gene expression levels relative to the internal reference gene (ß-actin) of the analyzed genes are listed in Table 3.
Gene Expression Levels of Cox-2, CCND1, EGFR, IL-8, and VEGF, and Response to Cetuximab The only factor that showed a significant correlation between response and gene expression level was VEGF (Table 4). Patients with a PR had a median gene expression level of 4.63 x 10–3; patients with stable disease, 3.76 x 10–3; and patients with progressive disease, 6.56 x 10–3 (P = .038, Kruskal-Wallis test).
Gene Expression Levels of Cox-2, CCND1, EGFR, IL-8, and VEGF, and Survival Gene expression cutoff values that best segregated patients into poor- and good-prognosis subgroups (in terms of likelihood of surviving) were defined for Cox-2, CCND1, EGFR, IL-8, and VEGF by using the maximal  2 method of Miller, Siegmund, and Halpern. The log-rank test was used to evaluate the association between gene expression levels and survival for each single gene. Using an EGFR cutoff value of 1.19 x 10–3, 21 patients had a low EGFR expression level, and nine had a high EGFR expression level. The median survival was 7.3 months (95% CI, 4.4 to 13.5 months) in patients with low EGFR mRNA levels, and 2.2 months (95% CI, 1.7 to 4.5 months) in patients with high EGFR mRNA levels (P = .09, log-rank test; Table 5 and Fig 2). The association between expression levels of CCND1, EGFR, IL-8, and VEGF, and survival did not show significant results or relevant trends, as indicated in Table 4.
The combination of low expression levels of Cox-2, EGFR, and IL-8 had a median overall survival of 13.5 months (95% CI, 5.5 to 15.9 months), and patients with high gene expression levels of these three genes had a median overall survival of 2.3 months (95% CI, 2.1 to 4.8 months; P = .028, log-rank test; Table 5 and Fig 3). In addition, the combination of gene expression levels of Cox-2, EGFR, and IL-8 was an independent prognostic factor after adjusting skin toxicity, which was associated with survival. Other combinations of genes did not show a significant relation to survival.
Gene Expression Levels of Cox-2, CCND1, EGFR, IL-8, and VEGF, and Skin Toxicity CCND1, IL-8, and VEGF gene expression levels did not show a significant correlation with the grade of skin toxicity. Patients with a grade 2 to 3 skin reaction had lower intratumoral EGFR gene expression levels compared with patients with a grade 0 to 1 toxicity; however, it did not reach statistical significance (0.85 v 1.19 x 10–3; P = .086). Patients with skin toxicity of higher grade had statistically significant lower gene expression levels of Cox-2 (0.27 v 1.20; P = .009; Table 4).
Skin Toxicity: A Surrogate for Response and Survival
IHC Analysis of EGFR in Tumor Samples IHC analysis of EGFR protein expression in tumor samples demonstrated the following: 23 (59%) had a weak EGFR staining (1+ intensity), 11 (28%) a moderate EGFR staining (2+ intensity), and five (13%) a strong EGFR staining (3+ intensity). There was no association of EGFR staining with response, survival, and toxicity (Table 7). In 30 patients, we were able to compare IHC and gene expression levels of EGFR (Table 8). We did not find a significant association.
The use of the EGFR targeting monoclonal antibody cetuximab in patients with metastatic CRC demonstrates promising efficacy in three different phase II clinical trials. However, there are currently no molecular markers established to identify patients most likely to benefit from this novel targeted therapy.2,12-14 Our goal was to identify gene expression levels of enzymes involved in the EGFR signaling pathway to predict response, survival, and toxicity in patients undergoing cetuximab treatment. Clinical trials investigating the effect of cetuximab in patients with metastatic CRC have failed to show a significant correlation between EGFR expression determined by IHC and clinical outcome.2,12 IHC is a semiquantitative and subjective method and is limited by the sensitivity of the monoclonal antibody and the tissue handling. However, in these trials, the presence of any skin toxicity was significantly associated with response and overall survival, suggesting that skin toxicity may be a surrogate marker. In our study, we confirmed the significant correlation between skin toxicity and time to tumor progression and overall survival. The association of skin toxicity and response did not reach statistical significance, probably due to the low number of responders in our study, but both patients who responded had a higher grade of skin toxicity. Using quantitative real-time PCR we were able to demonstrate that low intratumoral gene expression of VEGF was associated with response to cetuximab therapy, which was independent of skin toxicity. VEGF is one of the most important angiogenetic factors and is associated with poor prognosis and metastatic potential.21 This factor has been shown to be downstream of EGFR, and both pathways are linked through the neuropilin-1 receptor.22 Ciardiello et al23 demonstrated in GEO colon cancer cells a dose-dependent inhibition of VEGF with cetuximab therapy, and described in GEO xenografts in nude mice that cetuximab lead to a simultaneous reduction of VEGF expression and microvessel count. Furthermore, in vitro data suggest that VEGF is a main effector of EGFR to influence the regulation of angiogenesis in tumor development in glioblastoma and prostate cancer models.18,24 Therefore, one of the mechanisms of action of cetuximab is inhibition of angiogenesis, suggesting that high levels of intratumoral VEGF may lead to resistance to cetuximab treatment. We did not find a significant correlation between intratumoral EGFR gene expression and response, which may be because of the small sample size and low response rate. Therefore, the question remains unanswered whether a more accurate measurement of EGFR will be useful for predicting the response to therapy. Although EGFR gene expression levels did not correlate with response, patients in our study with higher gene expression of EGFR had a shorter overall survival. These findings are consistent with recent studies describing a significant association between increased protein levels of EGFR and poor prognosis in patients with CRC.25-27 Whether EGFR is not only a prognostic but a predictive factor is still unclear. Our study showed that patients with the combination of low gene expression levels of Cox-2, EGFR, and IL-8 showed a significantly longer overall survival that was independent of skin toxicity. These data suggest that Cox-2 and IL-8 may be important factors in the EGFR signaling pathway. Cox-2, a rate-limiting enzyme involved in the conversion of arachidonic acid to prostaglandins, is well known to affect tumorigenesis in the colon by the regulation of apoptosis, angiogenesis, and tumor cell invasiveness.28 Pai et al recently showed that prostaglandin E2, produced by the cyclo-oxygenase enzymes, transactivates EGFR in gastric epithelial and colon cancer cells, suggesting that Cox-2 induces the pathogenesis of cancer development by the activation of EGFR.15 In addition, Kinoshita et al29 described growth stimulation and induction of EGFR by the overexpression of cyclo-oxgenases in human colon cancer cells. IL-8, a member of the CXC chemokine family, was found to possess angiogenic and mitogenic properties. Overexpression of IL-8 is associated with increased tumor stage, disease progression, and metastatic potential in CRC.30 Perrotte et al17 showed that treatment with cetuximab in human transitional cell carcinoma growing orthotopically in nude mice inhibited mRNA and protein production of IL-8 accompanied by the involution of blood vessels. These data suggest that inhibition of angiogenesis by cetuximab may be by down-regulation of IL-8. Our findings implicate that Cox-2 may be an important regulator of EGFR, while IL-8 may play a part in cetuximab-related inhibition of angiogenesis. In addition, our data are consistent with studies describing Cox-2 and IL-8 as prognostic factors in different cancer types. High expression levels of Cox-2 are known to be associated with a shorter survival time in ovarian and head and neck cancer.28,31,32 Increased IL-8 expression has been associated with shortened overall survival in non–small-cell lung cancer.33 The most common side effect of cetuximab treatment is an acneiform follicular or perifollicular dermatitis that generally appears during the first 3 weeks of therapy.34 In our study, 85% of patients presented with such an acne-like rash. Interestingly, study patients with a grade 2 to 3 cutaneous reaction had significantly lower Cox-2 gene expression levels compared with patients who had grade 0 to 1 toxicity. Patients with lower EGFR mRNA amounts are more likely to have severe skin reactions compared with patients who have higher EGFR expression levels. These results imply that low levels of Cox-2 induce low levels of EGFR, leading to a down-regulated signaling pathway and therefore leading to the acne-like rash.
However, the relationship between Cox-2 and EGFR in the signaling pathway has been controversial (eg, Kulkarni et al showed that treatment with EGF markedly induced Cox-2 protein and Cox-2 mRNA, and stimulated Cox-2 promoter activity in cervical cancer cells).35 In lung cancer, it has been suggested that EGFR regulates Cox-2 by the transcription factor NF- We report for the first time that candidate genes (ie, Cox-2, EGFR, IL-8, and VEGF) are associated with clinical outcome independent of skin toxicity, suggesting that these members of the EGFR signaling pathway may play an important role in the efficacy of cetuximab. Our data are hypothesis-generating and should be validated in larger and prospective clinical trials.
The following authors or their immediate family members have indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. Employment: Kathleen D Danenberg, Response Genetics. Consultant/Advisory Role: Heinz-Josef Lenz, Chiron, Genentech, Response Genetics. Stock Ownership: Kathleen D. Danenberg, Response Genetics. Honoraria: Heinz-Josef Lenz, Eli Lilly, Pfizer, Roche, Sanofi. Research Funding: Heinz-Josef Lenz, NCI, NIH. For a detailed description of these categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and Disclosures of Potential Conflicts of Interest found in Information for Contributors in the front of each issue.
Supported by the National Institutes of Health grant 5 P30CA14089-27l, the San Pedro Guild Research Fund, and the Phase One Foundation Fund. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
INTRODUCTION
B.
