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FCGR2A and FCGR3A Polymorphisms Associated With Clinical Outcome of Epidermal Growth Factor Receptor–Expressing Metastatic Colorectal Cancer Patients Treated With Single-Agent Cetuximab

Wu Zhang, Michael Gordon, Anne M. Schultheis, Dong Yun Yang, Fumio Nagashima, Mizutomo Azuma, Heung-Moon Chang, Eva Borucka, Georg Lurje, Andy E. Sherrod, Syma Iqbal, Susan Groshen, Heinz-Josef Lenz

From the Division of Medical Oncology and the Departments of Pathology and Preventive Medicine, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, CA

Address reprint requests to Heinz-Josef Lenz, MD, FACP, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine, Sharon A. Carpenter Laboratory, 1441 Eastlake Ave, Suite 3456, Los Angeles, CA 90033; e-mail: lenz{at}usc.edu

	ABSTRACT

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Purpose Cetuximab, a chimeric immunoglobulin G 1 (IgG1) anti–epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), has shown efficacy in 10% of patients with metastatic colorectal cancer (CRC). Recent studies demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC) is one of the modes of action for rituximab and trastuzumab. Fragment c (Fc) portion of IgG1 mAb has shown to induce ADCC. Fragment c gamma receptors (FcR) play an important role in initiating ADCC. Studies have shown that two IgG FcR polymorphisms (FCGR2A-H131R and FCGR3A-V158F) independently predict response to rituximab in patients with follicular lymphoma. We tested the hypothesis of whether these two polymorphisms are associated with clinical outcome in metastatic CRC patients treated with single-agent cetuximab.

Patients and Methods Thirty-nine metastatic CRC patients were enrolled onto the ImClone0144 trial. Using an allele-specific polymerase chain reaction (PCR) –based method, gene polymorphisms of FCGA2A-H131R and FCGA3A-V158F were assessed from genomic DNA extracted from peripheral blood samples.

Results FCGR2A-H131R and FCGR3A-V158F polymorphisms were independently associated with progression-free survival (PFS; P = .037 and .055, respectively; log-rank test). Combined analysis of these two polymorphisms showed that patients with the favorable genotypes (FCGR2A, any histidine allele, and FCGR3A, any phenylalanine allele) showed a median PFS of 3.7 months (95% CI, 2.4 to 4.4 months), whereas patients with any two unfavorable genotypes (FCGR2A arginine/arginine or valine/valine) had a PFS of 1.1 months (95% CI, 1.0 to 1.4 months; P = .004; log-rank test).

Conclusion Our preliminary data suggest that these two polymorphisms may be useful molecular markers to predict clinical outcome in metastatic CRC patients treated with cetuximab and that they may indicate a role of ADCC of cetuximab.

	INTRODUCTION

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Colorectal cancer (CRC) represents the second most lethal malignancy in the United States. In 2006, it was projected that 148,610 new cases would be diagnosed and 55,170 deaths would occur.¹ Epidermal growth factor receptor (EGFR), a 170-kd transmembrane phosphoglycoprotein, is overexpressed in 70% to 80% of colorectal cancer patients, and overexpression of EGFR has been associated with tumor aggressiveness and poor prognosis.² Cetuximab, a chimeric immunoglobulin G 1 (IgG1) anti-EGFR monoclonal antibody (mAb), has shown promising efficacy in patients with metastatic CRC in several phase II clinical trials.^3,4 These studies led to US Food and Drug Administration approval of cetuximab in February 2004, when used in combination with irinotecan or alone, to treat patients with EGFR-expressing, metastatic CRC.

So far, there are no reliable markers that predict cetuximab efficacy. Phase II clinical trials by Cunningham et al³ and Saltz et al⁴ have failed to show a significant correlation between EGFR-staining intensity and patients' response to cetuximab treatment. Therefore, identifying molecular markers that can select patients who are likely to benefit from treatment of cetuximab is crucial to avoid chemotherapy toxicity and reduce treatment cost.

Cetuximab may exert its antitumor effects through multiple mechanisms. Cetuximab can competitively inhibit the binding of EGFR natural ligands, such as epidermal growth factor (EGF) and transforming growth factor alpha (TGF-), preventing receptor dimerization, phosphorylation, and downstream processes. As a result, the binding of cetuximab inhibits cell proliferation and angiogenesis, induces apoptosis of cancer cells, and prevents metastasis.^5,6 Another possible mechanism of its antitumor effects is through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is an immune effector mechanism in which antigen-specific antibodies direct immune effector cells of the innate immunity to kill the antigen–expressing cancer cells. In an in vitro study, less complete tumor growth inhibition was observed in A431 cancer cells with 225 F(ab')2 compared with the bivalent 225 mAbs. This finding suggests that in addition to ligand/receptor blockade, the immune mechanism also may contribute to the antitumor activity of intact 225 mAbs.⁷ As a chimeric IgG1 mAb, cetuximab also may produce cytotoxicity through immunologic mechanisms specific to Ig subtypes.^8,9

ADCC, via fragment c receptor (FcR) –bearing immune effector cells, plays a vital role in the antitumor effects of two other IgG1 mAbs: trastuzumab and rituximab. Several studies have shown that part of the antitumor effect of trastuzumab, a human IgG1 anti–human epidermal growth factor receptor 2 (HER-2) antibody, is through ADCC.¹⁰ Rituximab is a chimeric IgG1 mAb for B-cell differentiation antigen CD20. Several in vitro studies have shown similar results to those described for trastuzumab, suggesting that FcR-bearing ImClone immune effector cells mediated by ADCC play a predominant role in the antitumor efficacy of rituximab.^11,12

Recently, two functional FCGR gene polymorphisms have been identified that may affect the killing function of immune effector cells. Several studies have correlated these polymorphisms with clinical efficacy of rituximab. Cartron et al¹³ reported that FCGR3A-V158F polymorphism was associated with tumor response in follicular non-Hodgkin's lymphoma patients with first-line rituximab treatment. In vitro studies also found FCGR3A-158V allele has a higher affinity to human IgG1 than does the phenylalanine (F) allele. Cells bearing the FCGR3A-158V allele mediate ADCC more effectively.^14,15 Similarly, Weng et al¹⁶ found FCGR2A-H131R polymorphism is an independent predictor for rituximab response, with in vitro studies also showing the high-affinity 131H allele has higher binding efficiency for human IgG2 antibodies when compared with 131R allele.

The goal of our study was to explore the potential of FCGR2A-H131R and FCGR3A-V158F polymorphisms to serve as molecular markers that predict cetuximab response, overall survival (OS), and toxicity in metastatic CRC patients. We tested the hypothesis whether these two fragment c gamma receptor (FcR) gene polymorphisms, alone or in combination, are associated with response, survival, and toxicity in patients with metastatic CRC treated with cetuximab.

	PATIENTS AND METHODS

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Eligible Subjects
Thirty-nine patients with histopathologically confirmed metastatic CRC, who either failed at least two prior chemotherapy regimens or failed adjuvant therapy plus one chemotherapy regimen for metastatic disease (provided the patient progressed within 6 months of completing adjuvant therapy), were included in this study. We enrolled all 39 patients treated in our institution from October 2002 to March 2003 at the University of Southern California/Norris Comprehensive Cancer Center (USC/NCCC, Los Angeles, CA). These 39 patients were part of the phase II open-label multicenter study (ImClone [IMCL] trial 0144) of cetuximab (C225), which included a total of 346 patients. Our patient group has a similar median progression-free survival (PFS) of 2.4 months (95% CI, 1.4 to 3.7 months) and OS of 5.5 months (95% CI, 2.7 to 8.7 months) compared with this recently published phase II study (IMCL-0144),¹⁷ which has a median PFS of 1.4 months (95% CI, 1.4 to 2.1 months) and OS of 6.6 months (95% CI, 5.6 to 7.6 months).

Our study was conducted at USC/NCCC and was approved by the institutional review board at the University of Southern California for Medical Sciences (Los Angeles, CA). All patients showed immunohistochemical evidence of EGFR expression in their tumor samples. All patients signed an informed consent for tissue and blood collection for the study of molecular correlates. Blood samples were collected before initiation of chemotherapy.

Clinical Evaluation and Response Criteria
For patients with measurable disease, response was assessed every 6 weeks during the course of this study. An objective response was classified as a reduction of at least 50% tumor burden on computed tomography. All objective responses were required to be confirmed by a follow-up scan at least 4 weeks following documentation of the response. Tumor progression on study was defined as an increase of at least 25% in the overall area of the tumor, or the appearance of new lesions. Response to cetuximab was also evaluated retrospectively by an independent response assessment committee that was blinded to the investigator-reported measurements and assessments. Patients underwent weekly blood counts, and physical examinations were performed every third week while on study.

Patients were infused with cetuximab at standard loading dose 400 mg/m² over a 2-hour period, followed by a weekly infusion of 250 mg/m² treatment over a 1-hour period. Treatment was continued until progression of disease or toxicity occurred, and patients were evaluated every 6 weeks for tumor response.

FCGR2A-H131R and FCGR3A-V158F Genotyping
A peripheral blood sample was collected from each patient, and genomic DNA was extracted from WBCs using the QiaAmp kit (Qiagen, Valencia, CA). FCGR2A-H131R polymorphism was tested by PCR, followed by allele-specific restriction enzyme digestion method, as previously described.¹⁸ Briefly, forward primer 5'-GGAAAATCCCAGAAATTCTCGC-3' and reverse primer 5'-CAACAGCCTGACTACCTATTACGCGGG-3' were used for PCR amplification, and annealing temperature was 55°C. PCR product was digested with 20 units BstUI restriction enzyme (New England Biolabs, Beverly, MD), and alleles were separated on 3% NuSieve ethidium bromide-stained agarose gel (FMC BioProducts, Ipswich, MA). The FCGR2A-131H and FCGR3A-131R alleles produced DNA fragments of 343 base pair and 322 base pair, respectively.

For FCGR3A-V158F polymorphism, allele-specific PCR was amplified using each allele-specific forward primer 5'-CTGAAGACACATTTTTACTCCCAAA/C-3' and reverse primer 5'-TCCAAAAGCCACACTCAAAGAC-3'. The annealing temperature was 64°C. The reaction products were run on 4% NuSieve ethidium bromide-stained agarose gel. Seventy-three base pair PCR fragment either positive for valine (V) or F allele was visualized under UV light as previously described.¹⁹

Statistical Analysis
OS was the primary end point in the analysis. Objective tumor response, toxicity (acne-like rash), and PFS were the secondary outcome variables. The OS time was calculated as the period from the first day of cetuximab infusion until death from any cause, or until date of last follow-up, at which point data were censored. PFS was calculated as the time for the first day of cetuximab treatment until the first observation of disease progression or death from any cause. If a patient had not progressed or died, PFS was censored at the time of last follow-up.

The association of each polymorphism with OS and PFS was analyzed using Kaplan-Meier curves and the log-rank test. In the univariate analyses, the Pike estimate of relative risk with 95% CI was based on the log-rank test.²⁰ The associations of each polymorphism with tumor response and toxicity were summarized using contingency tables and the exact conditional test^21,22 for tumor response and Fisher's exact test for toxicity. The distributions of polymorphisms across demographic characteristics were examined using Fisher's exact test. Finally, the Cox proportional hazards regression model with stratification factors was fitted to re-evaluate the association among FCGR polymorphisms, and PFS, and OS, considering the imbalances in the distributions of baseline characteristics. P values of the log-likelihood ratio test were obtained from the modeling.

All tests of statistical significance were two-sided. The analyses were performed using the SAS statistical package version 9.0 (SAS Institute Inc, Cary, NC) and Epilog Plus version 1.0 (Epicenter Software, Pasadena, CA).

	RESULTS

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Thirty-nine patients were enrolled in 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 for association between EGFR expression (detected by immunohistochemical staining) and clinical outcome. Out of 39 patients, 35 patients' samples were available for molecular tests. At the time of analysis, two patients were still alive, and the follow-up time for those two patients was 2.5 and 3.7 months. The median survival time was 5.5 months (95% CI, 2.7 to 8.7 months). Under cetuximab treatment, two patients (6%) had partial response (PR), 21 (60%) had stable disease (SD), and 12 (34%) had progressive disease (PD), whereas no patient showed complete response. Four patients were not assessable for response. Skin reactions were observed in 85% of the 39 patients, where 12 patients (31%) had a grade 1 reaction, 20 (51%) patients had a grade 2 reaction, and one patient (3%) had a grade 3 reaction (Table 1). Baseline patient characteristics (Table 1) were not statistically and significantly associated with clinical outcome (response, rash, PFS, and OS).

Combination Analysis
Combination analysis resulted in a highly statistically significant relationship between these two polymorphisms and PFS (P = .004). Patients with two favorable alleles (FCGR2A, any histidine [H] allele, and FCGR3A, any F allele) had a median PFS time of 3.7 months (95% CI, 2.4 to 4.4 months), whereas patients with any two unfavorable genotypes (FCGR2A R/R or FCGR3A V/V) survived 1.1 months (95% CI, 1.0 to 1.4 months; Fig 1). Combination analysis of these two FCGR polymorphisms for response showed that patients with two favorable alleles had improved response when compared with any two unfavorable alleles (P = .003, exact conditional test; Table 2).

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Progression-free survival for patients with metastatic colorectal cancer receiving cetuximab by fragment c receptor (FCGR) polymorphisms. H, histidine allele; R, arginine allele; F, phenylalanine allele; V, valine allele.

	DISCUSSION

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One of the most important mechanisms of cetuximab is through inhibition of receptor/ligand interaction of the EGFR-signaling pathway. Moroni et al²³ found that EGFR gene copy number may predict patients' response to the anti-EGFR mAbs, cetuximab, and panitumumab. They assessed EGFR gene copy number of 31 colorectal cancer patients receiving either cetuximab or panitumumab treatment by florescence in situ hybridization (FISH) technique. Eight of nine responders had an increased EGFR copy number, whereas only one of 21 nonresponders had increased EGFR copy number. In another study, Lievre et al²⁴ screened tumors from 30 metastatic colorectal cancer patients treated with cetuximab for KRAS, BRAF and PIK3CA mutation. They found KRAS mutation was significantly associated with resistance to cetuximab. Our group has previously tested the key gene polymorphisms in the EGFR-signaling pathway. We demonstrated that cyclinD1 and EGF gene polymorphisms are significantly associated with cetuximab efficacy in the same population as this study.²⁵ These studies all point out that patients' genetic makeup may affect the sensitivity of their tumors to cetuximab.

The other pathway through which cetuximab may exert its antitumor effect is ADCC. Several in vitro and in vivo studies have shown that as a chimeric IgG1 mAb, cetuximab binds to the antigen on the surface of tumor cells and its Fc portion binds to the immune effector cells through FcR. Consequently, this binding activates the immune effector cells, leading to tumor cell killing.^7,8 Studies for trastuzumab and rituximab also confirmed that ADCC is the predominant mechanism of tumor killing.^26,27 In ADCC, mAb-attacking tumor cells are mediated by immune effector cells that express Fc Receptor. Recently, two FcR gene polymorphisms have been identified that affect the binding of IgG, changing ADCC function and affecting clinical tumor response. FCGR2A-H131R polymorphism is located at the extracellular ligand-binding domain. It either has an H or arginine (R) allele at amino acid position 131. The FCGR2A-131H/H genotype was shown to have a higher affinity to human IgG2 in an in vitro study. FCGR2A-131H/R polymorphism is an independent predictor for patient's response to rituximab treatment.^16,28 The other well-studied polymorphism is FCGR3A-V158F polymorphism, which encodes either a V or F at amino acid position 158. In vitro studies have shown FCGR3A V allele has a higher binding affinity to human IgG1 than the F allele, indicating immune effector cells bearing FCGR3A V allele mediate ADCC more effectively.¹⁵ Clinical studies from Weng et al¹⁶ and Cartron et al¹³ have shown FCGR3A-158V/V genotype is associated with better response rate and longer remission in patients with follicular lymphoma treated with rituximab.

Our study found patients with FCGR2A-131H allele had a longer PFS than patients with FCGR2A-131R allele when treated with single-agent cetuximab. This finding is in accordance with a previous study on rituximab by Weng et al,¹⁶ which also showed that FCGR2A-131H/H genotype is associated with higher response rate and longer remission than FCGR2A-131H/R and R/R genotypes. The mechanism to explain FCGR2A-H131R polymorphism and cetuximab efficacy is still unclear. Previous in vitro studies have demonstrated that FCGR2A-131H allele binds to human IgG2 better than 131R allele, but 131H allele also shows low affinity binding to murine IgG1. Also, no significantly different binding affinity was observed between these two alleles and binding of human IgG1.²⁹ In addition, Shields et al¹⁴ studied the binding site on human IgG1 for FCGR2A; they found that a certain class of variants of human IgG1–binding sites may also improve or reduce its binding to FCGR2A.

Several clinical studies also report controversial results regarding the role of FCGR2A-H131R polymorphism in different cancer patients. Lin et al³⁰ report that FCGR2A-H131R polymorphism did not predict response to alemtuzumab, a humanized IgG1 anti-CD52 mAb, in patients with chronic lymphocytic leukemia. Meanwhile, Cheung et al³¹ found FCGR2A-131R/R genotype, instead of 131 H/H genotype, is correlated with better clinical outcome in neuroblastoma patients treated with a murine IgG3 anti-GD2 mAb 3F8. The discrepancy in these studies may be due to different Ig type (IgG1vs IgG3, human versus murine). Further in vitro and in vivo studies are necessary to elucidate the exact relationship between FCGR2A-H131R polymorphism and cetuximab efficacy.

Our study found FCGR3A-158V/V genotype is associated with shorter PFS compared with patients with 158V/F or F/F genotype. This is against our hypothesis and the findings in the NHL patients treated with rituximab, which showed that patients with FCGR3A-158V/V genotype are associated with higher response rate and longer PFS. One explanation of this conflicting result may be like FCGR2A, a certain class of variants of human IgG1–binding sites may also improve or reduce its binding to FCGR3A. Another possible explanation is that recent studies have found that besides FCGR2A and FCGR3A, which are both activating FcRs, the inhibitory receptor FCGR2B also is a potent regulator of ADCC in vivo. Clynes et al¹² found trastuzumab and rituximab engages both activatory (FCGR2A and FCGR3A) and inhibitory receptors (FCGR2B), and the in vivo activity of these IgG1 antibodies may more be predictable by the ratio of FCGR3A to FCGR2B (so-called A/I ratio). Interestingly, studies from Farag et al³² and Lin et al³⁰ also showed that FCGR 158 V/V genotype, which is associated with high-affinity binding to IgG, correlated with low response rate to rituximab and alemtuzumab in chronic lymphocytic leukemia, respectively. The exact role of FCGR3A-V158F polymorphism and cetuximab efficacy require more basic studies to determine its relevance in predicting clinical outcome of cetuximab.

To conclude, our data suggest that two FCGR polymorphisms have significant association with PFS and response in metastatic CRC patients treated with single-agent cetuximab. However, our findings present limitations, and results should be interpreted cautiously. The first of these limitations is the fact that our study is based on a small number of patients (N = 35) treated in a single institution. There is still debate whether ADCC plays a role in metastatic cancer patients that mostly have suppressed immune function. For this reason, early-stage CRC patients treated with cetuximab in the adjuvant setting may be more suitable study candidates. Nevertheless, these limitations could not outweigh the importance of this pilot study and our findings should be confirmed in larger, prospective trials.

	AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Employment or Leadership Position: None Consultant or Advisory Role: Heinz-Josef Lenz, Chiron, Genentech, Response Genetics Stock Ownership: None Honoraria: Heinz-Josef Lenz, Eli Lilly & Co, Pfizer, Roche, Sanofi-aventis Research Funding: Heinz-Josef Lenz, National Cancer Institute, National Institutes of Health Expert Testimony: None Other Remuneration: None

	AUTHOR CONTRIBUTIONS

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Conception and design: Wu Zhang, Michael Gordon, Georg Lurje, Syma Iqbal, Susan Groshen, Heinz-Josef Lenz

Financial support: Heinz-Josef Lenz

Provision of study materials or patients: Fumio Nagashima, Mizutomo Azuma, Heung-Moon Chang, Andy E. Sherrod, Heinz-Josef Lenz

Collection and assembly of data: Wu Zhang, Anne M. Schultheis, Fumio Nagashima, Mizutomo Azuma, Heung-Moon Chang, Eva Borucka, Susan Groshen

Data analysis and interpretation: Dong Yun Yang, Georg Lurje

Manuscript writing: Wu Zhang, Michael Gordon

Final approval of manuscript: Wu Zhang, Dong Yun Yang, Georg Lurje, Syma Iqbal, Susan Groshen, Heinz-Josef Lenz

	NOTES

 
Supported by the National Institutes of Health Grants No. 5 P30CA14089-271, San Pedro Guild Research Fund, and the Dhont Family Foundation.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted August 25, 2006; accepted May 29, 2007.

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