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Journal of Clinical Oncology, Vol 24, No 30 (October 20), 2006: pp. 4914-4921 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.06.7595 Multicenter Phase II and Translational Study of Cetuximab in Metastatic Colorectal Carcinoma Refractory to Irinotecan, Oxaliplatin, and Fluoropyrimidines
From the Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA; University Hospital Gasthuisberg, Leuven, Belgium; Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ; Dana-Farber/Partners CancerCare, Boston, MA; Swedish Cancer Institute, Seattle, WA; St Joseph Mercy Hospital, Ann Arbor, MI; Center for Oncology Research and Treatment, Dallas, TX; Rocky Mountain Cancer Center, Denver, CO; Lakeland Regional Cancer Center, Lakeland, FL; and ImClone Systems Incorporated, New York, NY Address reprint requests to Eric K. Rowinsky, MD, ImClone Systems, 33 ImClone Dr, Branchburg, NJ 08876; e-mail: eric.rowinsky{at}imclone.com
PURPOSE: This multicenter study evaluated the antitumor activity of cetuximab, an immunoglobulin G1 antibody directed at the epidermal growth factor receptor (EGFR), in metastatic colorectal carcinoma (CRC) refractory to irinotecan, oxaliplatin, and a fluoropyrimidine. It also evaluated the safety, pharmacokinetics, immunokinetics, and biologic determinants of activity. PATIENTS AND METHODS: Patients with metastatic CRC, whose tumors demonstrated EGFR immunostaining and were refractory to irinotecan, oxaliplatin, and fluoropyrimidines, were treated with cetuximab at a loading dose of 400 mg/m2 followed by 250 mg/m2 weekly. An independent review committee (IRC) reviewed responses. Blood was collected for cetuximab pharmacokinetics and to detect antibodies to cetuximab. EGFR gene sequencing of the tyrosine kinase domain and gene copy number assessments were performed. RESULTS: The response rates in 346 patients, as determined by the investigators and IRC, were 12.4% (95% CI, 9.1 to 16.4) and 11.6% (95% CI, 8.4 to 16.4). The median progression-free survival (PFS) and survival times were 1.4 months (95% CI, 1.4 to 2.1) and 6.6 months (95% CI, 5.6 to 7.6), respectively. An acneiform rash occurred in 82.9% of patients; grade 3 rash was observed in 4.9%. Response and survival related strongly to the severity of the rash. In contrast, clinical benefit did not relate to EGFR immunostaining. EGFR tyrosine kinase domain mutations were not identified, and EGFR gene copy number did not relate to response or PFS, but to survival (P = .03). CONCLUSION: Cetuximab is active and well tolerated in metastatic CRC refractory to irinotecan, oxaliplatin, and fluoropyrimidines. The severity of rash was related to efficacy. Neither EGFR kinase domain mutations nor EGFR gene amplification appear to be essential for response to cetuximab in this setting.
The selection of epidermal growth factor receptor (EGFR) as a target in the therapy of colorectal cancer (CRC) reflects its ubiquitous expression and association with adverse biologic features.1-7 Furthermore, monoclonal antibodies and small molecule tyrosine kinase (TK) inhibitors of EGFR are active in well established human cancers in preclinical studies.8-11 Cetuximab (Erbitux; ImClone Systems Inc, New York, NY), an immunoglobulin G1 (IgG1) monoclonal antibody to the ligand-binding domain of EGFR, inhibits receptor activation by interfering with binding of stimulatory ligands.4-9,11-13 Cetuximab binding results in robust receptor internalization and reduced numbers of receptors.4,5,7,12,13 By blocking downstream signaling, cetuximab inhibits cell proliferation, angiogenesis, and metastasis, and promotes apoptosis.4,5,7,12-20 Being an IgG1 construct, it also engages immune effector cells that mediate antibody-dependent cell-mediated cytotoxicity.4,5,21 The most impressive activity with cetuximab in preclinical studies has been in combination with various cytotoxic modalities, even in resistant tumors.4-7,12,22-25 Therefore, cetuximab's early development focused on evaluating the merits of combining cetuximab and irinotecan in irinotecan-refractory CRC.26 When 329 irinotecan-refractory CRC patients were randomly assigned to treatment with either cetuximab plus irinotecan or cetuximab alone, response rates were 22.9% and 10.8%, and median times to progression were 4.1 and 1.5 months, respectively. Survival did not significantly differ (median values, 8.6 and 6.9 months, respectively).27 In another study, 9% of 57 irinotecan-refractory CRC patients responded to cetuximab monotherapy.28 At the time of the aforementioned trials, oxaliplatin had not yet undergone regulatory approval in the United States or incorporation into treatment schemes worldwide. The primary objective of this study was to determine the activity of cetuximab in metastatic CRC refractory to both irinotecan and oxaliplatin, with information obtained on the toxicity, safety, and pharmacokinetics of cetuximab, and to relate EGFR gene mutations and gene amplification to therapeutic results.
Patient Eligibility Patients with pathologically confirmed CRC and any degree of EGFR immunostaining as performed at Impath (Los Angeles, CA) were eligible. EGFR progression must have been documented during or within 3 months after treatment with irinotecan, oxaliplatin, and fluoropyrimidines in the metastatic setting or within 6 months of adjuvant therapy, with at least two chemotherapy regimens for metastatic disease or adjuvant therapy plus at least one regimen for metastatic disease. The extent of prior chemotherapy was not restricted. Eligibility criteria also included: age  18 years, Eastern Cooperative Oncology Group performance status of 0 or 1; life expectancy of at least 3 months; no major surgery, radiation, chemotherapy, or investigational agent within 4 weeks; normal hematopoietic, hepatic, and renal functions; measurable disease; no coexisting medical problem of sufficient severity to limit study compliance; no history of brain metastases; and no prior treatment with EGFR-targeting agents or murine or chimeric antibodies. Patients gave written informed consent before treatment and evaluation of tumor samples.
Dosage and Drug Administration Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria (version 2.0). Treatment modifications were indicated for hypersensitivity reactions (HSRs) and severe skin toxicity. For HSRs of grade 1 severity, the infusion rate was reduced by 50%. For grade 2 HSRs, cetuximab was discontinued and symptomatic measures undertaken until the toxicity resolved to grade 1 or less, at which time cetuximab was resumed at 50% of the initial rate. Cetuximab was discontinued for grade 3 or 4 HSRs. For skin toxicity of grade 3 severity, cetuximab was discontinued until the toxicity resolved to grade 2 or less, at which time it was resumed. If the toxicity did not resolve to at least grade 2 within 4 weeks or grade 3 toxicity recurred at least four times, cetuximab was discontinued. A second or third recurrence of grade 3 toxicity required dose reductions to 200 and 150 mg/m2.
Pretreatment and Follow-Up Studies Tumors were measured pretreatment and every 6 weeks and criteria for tumor response were based on modified WHO guidelines.29,30 A partial response (PR) required at least a 50% reduction in the sum of the bidimensional products of all measurable lesions documented at least 4 weeks apart. An independent review committee confirmed all responses. Treatment was continued in the absence of intolerable toxicity or progressive disease, defined as at least a 25% increase in measurable disease, unequivocal growth of existing nonmeasurable disease, the appearance of one or more new lesions, or reappearance of old lesions.
Pharmacokinetic and Immunologic Studies A double-antigen radiometric assay was performed to measure anticetuximab antibodies.31 We sampled blood (5 mL) before the first cetuximab infusion; at weeks 6, 12, 18, 24, and 30; and during the follow-up visit. A positive response was defined as two consecutive postdose measurements of anticetuximab antibody levels exceeding the upper limits of normal from untreated subjects (> 7 ng/mL of cetuximab binding) and at least twice the pretreatment value.
EGFR Gene Sequencing and Amplification Studies
Statistical Analyses
Delta Ct values were related to best response using nonparametric analysis of variance. Proportional hazards (Cox) modeling was used to relate
General EGFR immunostaining was positive in the tumors of 480 (93%) of 516 patients screened, and 346 patients (67%; Table 1) who fulfilled all eligibility criteria and received treatment. On subsequent review, EGFR was not detected in the tumors of nine (2.6%) patients. At the time of analysis, 344 patients (99%) had discontinued treatment, most commonly for disease progression (317 patients or 92%). Other reasons included adverse events (12 patients or 3.5%), withdrawal of consent (four patients or 1.1%), death (seven patients or 2.0%), unknown cause (two patients or 0.6%), intestinal perforation (one patient or 0.3%), noncompliance (one patient or 0.3%), and other reasons unrelated to study drug and disease (three patients or 1.0%).
Patients received a median of four prior chemotherapy regimens. All patients had been treated with both irinotecan- and oxaliplatin-based regimens, with 93.6% and 98.3% developing progressive disease during treatment or within 3 months of treatment with these agents, respectively, in the metastatic setting. Sixty-six percent, 63%, and 17% of patients most recently received fluoropyrimidine-, oxaliplatin-, and irinotecan-containing regimens, respectively. Ninety-one percent and 80% discontinued their most recent irinotecan- and oxaliplatin-containing regimens for tumor progression, respectively, at a median of 0.5 months after their last treatment. The best response to the most recent irinotecan-based regimen was: 2.0% complete response, 13% partial response (PR), 49% stable disease, and 31% progressive disease; the best response to the most recent oxaliplatin-based regimen was 0.6% complete response, 5.8% PR, 39.3% stable disease, and 49.7% progressive disease. The median duration of cetuximab treatment was 9 weeks (range, 1.0 to 66.3 weeks), with a median of nine infusions per patient (range, 1 to 56 infusions). The median dose intensity was 249 mg/m2/wk. Most patients (97%) did not require dose reduction.
Antitumor Activity
Neither sex, race, age, site of primary malignancy, performance status, nor EGFR immunostaining related to response (Table 3). Of the nine patients whose tumors had no EGFR immunostaining, one patient (11.1%) had a PR and three had stable disease. Except for Eastern Cooperative Oncology Group performance status 0 or 1 (median survival, 9.0 months [95% CI, 8 to 11.2] v 4.8 months [95% CI, 3.7 to 6.2]; P = .02), survival did not relate to the other variables.
The presence and severity of rash related to clinical benefit (Table 3). Forty (13%; 95% CI, 9.3 to 17.1%) of 311 patients who developed rash experienced a PR, whereas none of the 35 patients without rash responded. PRs occurred in 7%, 17%, and 20% of patients with grade 1, 2, or 3 rashes, respectively. Overall survival correlated strongly with the presence and severity of the rash, with median survival times of 1.7 months (95% CI, 1.2 to 2.3) for patients without rash, and 4.9 months (95% CI, 3.6 to 6.6) and 9.4 months (95% CI, 8.1 to 11.0) for those with grade 1 and 2-3 rash, respectively (Fig 3).
Toxicity Toxicities related to cetuximab that occurred in at least 10% of patients are presented in Table 4. The characteristic rash, often described as acne, maculopapular, pustular, dry skin, or exfoliative dermatitis, occurred in 90% of patients. The median time to the first onset of rash was 8 days, whereas the median time to grade 3 toxicity was 19 days. Other common adverse events attributed, in part, to cetuximab are detailed in Table 4. Twelve patients (3.4%) discontinued cetuximab because of adverse events including one for rash (grade 2). Decrements in left ventricular ejection fraction were not observed.
HSRs were experienced by 26 (7.5%) patients during or shortly after the first cetuximab dose, but only six (1.7%) patients had serious reactions. Five of these subjects received premedication with an H1-histamine antagonist before cetuximab treatment. One patient who had a serious HSR following premedication experienced no further toxicity after re-treatment on several occasions preceded by premedication with corticosteroids and diphenhydramine.
Pharmacokinetics and Immunokinetics Eight (4.26%) of 188 patients who had blood sampled pre- and post-treatment developed anticetuximab antibodies, which did not impact on pharmacokinetics or relate to antitumor activity.
EGFR Sequencing and Amplification Studies
EGFR gene copy number was determined in the colorectal tumor samples of all 34 patients from whom adequate tissue and informed consent could be obtained. Eight patients (23.5%) in this group had PRs. Scatterplots depicting relationships between
In this study, cetuximab monotherapy produced notable antitumor activity in metastatic CRC refractory to irinotecan, oxaliplatin, and a fluorpyrimidine, confirming the results of previous studies but in a more refractory setting.26-28 In prior phase II trials, response rates of 9.0% and 10.8% were reported, respectively,26-28 with cetuximab-based therapy similarly effective in irinotecan-refractory patients regardless of whether they had received prior oxaliplatin.27 The activity of cetuximab in the present study is also similar to that of irinotecan-based treatment, and exceeded that of oxaliplatin-based treatment in patients who received these regimens before cetuximab. These findings suggest negligible, if any, cross-resistance between cetuximab and either irinotecan and oxaliplatin. Antitumor activity in irinotecan and oxaliplatin-refractory CRC comparable to that of cetuximab has not been reported with other therapeutics. Still, although the activity of cetuximab monotherapy is notable, cetuximab-based combinations have conferred greater benefit,26-28 with two-fold higher response rates and nearly three-fold longer PFS with cetuximab plus irinotecan compared with cetuximab alone, even in irinotecan-refractory patients.27 Cetuximab monotherapy should be reserved for patients who cannot tolerate combination therapy. Activity was also noted in patients whose tumors had no EGFR immunostaining, confirming prior reports, and likely due to the pitfalls of immunohistochemistry in assessing EGFR expression.26,38 While rash was disturbing from an esthetic standpoint, it was rarely severe or resulted in termination of treatment. Consistent with other reports of both EGFR-targeting antibodies and receptor TK inhibitors, the severity of the rash related strongly to both response and survival.39-42 In contrast, neither sex, race, age, site of primary malignancy, performance status, nor EGFR gene copy number related to clinical benefit. The effects of cetuximab in skin may reflect the extent of EGFR blockade in both patients and tumors. Although a plausible explanation for the relationship of rash and clinical benefit is that rash may relate to EGFR saturation or attainment of relevant concentrations or other pharmacologic parameters indicative of an EGFR effect, the severity of rash has been a stronger indicator of drug activity than other parameters.39-41 However, if rash is a surrogate of EGFR saturation in tumors, then exceeding threshold concentrations or titrating dose to a maximally tolerated severity of cutaneous toxicity may further increase the efficacy of cetuximab. This hypothesis is being tested in the EVEREST trial, in which patients with irinotecan-refractory CRC are randomly assigned to treatment with irinotecan plus cetuximab on either a conventional dose-schedule or titrated to a maximal-tolerated rash. However, the high incidence of skin toxicity at standard doses of EGFR-targeting therapeutics indicates that there is a narrow therapeutic dosing window. Alternate explanations for the concordance between activity and rash include inherent factors such as EGFR polymorphisms common to tumor and skin and immunocompetence. Cetuximab, an IgG1 construct, can mediate inflammatory and cytotoxic reactions, by inducing antibody-dependent cell-mediated cytotoxicity, which may contribute to the inflammatory reaction observed in skin rashes. EGFR TK domain mutations and gene copy number were related to outcome.43-45 None of the tumors sequenced for exons 18, 19, and 21 had any of the mutations associated with response to the EGFR TK inhibitors.43-45 Since the samples included tumors from 11 PRs, EGFR mutations do not seem to be required to derive benefit from cetuximab. Although mutations were initially noted in some samples, they were not found in the PCR products amplified independently from the original PCR products. Additionally, no mutations were identified previously in same regions of the EGFR TK domain sequenced from DNA of 160 fresh biopsies of CRC patients, indicating that EGFR mutations in these exons are rare in CRC.46-48 In contrast, similar mutations have been noted in CRC and lung cancer samples that underwent formalin fixation.49-52 Such mutations have also been detected in paraffin embedded DNA samples from normal tissues, suggesting that they are artifacts of fixation.53 Collectively, these findings suggest that the "irreproducible mutations" were not in the original tumors. EGFR gene copy number, as assessed by quantitative PCR, related to neither response nor PFS. In contrast, Moroni et al reported that EGFR gene copy number, measured by fluorescence in situ hybridization (FISH), relates to the propensity of CRC to respond to EGFR-directed antibodies.48 In another retrospective study involving non–small-cell lung cancer patients treated with EGFR TK inhibitors, EGFR gene copy number, as assessed by FISH also related to better response and longer PFS.54 The use of different techniques, PCR and FISH, may have contributed to the disparate results. Although FISH may be affected less by disomic tumor cells or stromal contaminants and may be superior in situations in which EGFR gene copy number is low,54 the quantitative PCR analysis in this study utilized valid controls and the results were reproducible. Despite the lack of relationships between EGFR gene copy number, response and PFS, survival appeared to relate to gene copy number. In patients with non–small-cell lung cancer, increased EGFR gene copy number detected by FISH, sometimes accompanied by a correspondingly higher EGFR protein expression, is associated with improved survival.54,55 This finding suggests that EGFR gene copy number is an independent prognostic variable, possibly relating to CRC biology and not necessarily predictive of the therapeutic outcome with cetuximab. Several other possible molecular determinants of benefit to cetuximab in CRC have also been reported. For example, higher vascular endothelial growth factor gene expression has been related to cetuximab resistance, whereas the combination of low gene expression levels of Cox-2, EGFR, and interleukin-8 was related to increased overall survival.56 KRAS mutations have also been related to cetuximab resistance and a worse prognosis.57 This study indicates that cetuximab monotherapy has notable activity in metastatic CRC refractory to irinotecan, oxaliplatin, and a fluoropyrimidine; however, cetuximab combined with relevant cytotoxics and other rationally designed therapeutics would be expected to confer additional benefit over monotherapy in drug refractory and less heavily pretreated patients with CRC, and randomized trials evaluating the merits of cetuximab combined with both cytotoxic and target-based therapeutics in earlier disease settings are underway.
The following investigators and centers participated in this multicenter trial: H.-J. Lenz, Norris Cancer Center, University of Southern California, Los Angeles; E. Van Cutsem, University Hospital, Gasthuisberg, Leuven, Belgium; R. Mayer, Dana-Farber Cancer Institute, Boston; P. Gold, Swedish Cancer Institute, Seattle; P. Stella, St Joseph Mercy Hospital, Ypsilanti, Michigan; B. Mirtsching, Center for Oncology Research, Dallas; A. Cohn, Rocky Mountain Cancer Center, Denver; A. Pippas, Lakeland Regional Cancer Center, Lakeland, Florida; D. Strickland, Memphis Cancer Center, Memphis; H. Hochster, New York University, New York; L. Zehngebot, Florida Hospital, Orlando; J. Gurtler, East Jefferson Specialty Center, Metairie, Louisiana; J. Wade, Cancer Care Specialists, Decatur, Illinois; E. Samuel, North Shore Hematology Oncology, East Setauket, New York; D. Brandt, Northwestern Connecticut Oncology, Torrington, Connecticut; P. Loehrer, Indiana University, Indianapolis; C. Henderson, Peachtree Hematology Oncology, Atlanta; A. Hageboutros, Cooper Cancer Institute, Camden, New Jersey; B. Rosenbloom, Tower Hematology Oncology, Los Angeles; Y. Humblet, University Hospital Saint Luc, Brussels, Belgium; H. Bleiberg, Institut Jules Bordet, Brussels, Belgium; J. Waples, Comprehensive Cancer Institute, Huntsville, Alabama; J. Ahlgren, George Washington Medical Center, Washington, DC; P. Eisenberg, California Cancer Care, Greenbrae, California; P. Kaywin, South Florida Oncology Hematology, Miami; J. Marshall, Georgetown University Medical Center, Washington, DC; K. Dicke, Arlington Cancer Center, Arlington, Texas; I. Oliff, Hematology Oncology Associates of Illinois, Skokie, Illinois; T. Pluard, Missouri Cancer Care, Saint Charles, Missouri; F. Coco, Cancer Center of Boston, Boston; P. Cobb, Northern Rockies Hematology Oncology, Billings, Montana; M. Levine, Greater Baltimore Medical Center, Baltimore; L. Rosen, Cancer Institute Medical Group, Santa Monica, California; H. Tezcan, North Idaho Cancer Center, Coeur d'Alene, Idaho; W.G Harker, Intermountain Hematology Oncology, Salt Lake City; A. Forero-Torres, University of Alabama, Birmingham, Alabama; N.S. Tchekmedyian, Pacific Shores Medical Group, Long Beach, California; I. Wiznitzer, Comprehensive Cancer Care, Boca Raton, Florida; J. Fuloria, Ochsner Clinic Foundation, New Orleans; M. Lee, Park Nicollet Oncology Research, Saint Louis Park, Minnesota.
Genomic DNA was isolated from 5-µm-thick sections of paraffin-embedded tumors fixed on glass slides, using QIAamp DNA Mini Kit (Qiagen, Valencia, CA). The paraffin was removed by extraction with xylene and the tissue sample was then processed according to the protocol provided with the kit. Polymerase chain reaction (PCR) primers were designed against the exons 18, 19, and 21 of human epidermal growth factor receptor (EGFR) gene, using Primer 3 software (Whitehead Institute, Cambridge, MA).32,33 Primers had a M13 phage derived forward sequence (5'-TGTAAAACGACGGCCAGT-3') or a reverse sequence (5'-CAGGAAACAGCTATGACC-3') attached at the 5' end, to allow sequencing of PCR products with universal forward and reverse sequencing primers as presented in Table A1. Each 25-µL PCR cocktail contained 15 ng genomic DNA, 2.5 mmol/L MgCl2, 300 µmol/L dNTPs, and 2.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA). PCR products were purified using solid phase reversible immobilization before use as templates in DNA sequencing reactions. Sequencing was performed using the dye-terminator chemistry.34,35 Reaction products were analyzed by capillary electrophoresis in a PRISM 3730 x DNA Analyzer (Applied Biosystems), and sequence traces were analyzed for mutations with PolyPhred software (University of Washington, Seattle, WA).36
After isolating DNA, a quantitative PCR assay was developed to measure the abundance of the EGFR gene relative to that of a control gene, VEST1.37 Reactions were performed in triplicate and contained 15 ng of genomic DNA, 100 nmol/L Taqman probe and 200 nmol/L primers. Thermal cycling conditions were 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Reactions were performed on an ABI 7900HT Sequence Detection System (Applied Biosystems) using absolute quantification of Ct (threshold cycle) values. DiFi and A431 cell lines were used as positive controls. Analysis of standard curves of serially diluted DNA indicated that Ct values and log (DNA concentration) were linearly related. The difference in Ct values between the triplicate average for the EGFR and VEST1 probes was calculated (
The author or immediate family members 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. 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.
Dollar Amount Codes (A) <$10,000 (B) $10,000-99,999 (C)
The authors are grateful to Christopher Harbison, William Geese, and John Feder for their efforts with mutation and gene copy number analyses.
Supported by ImClone Systems, New York, NY, and Merck, Darmstadt, Germany. The investigators and centers participating in this multicenter, multinational study are listed in online only Appendix 1. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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ABSTRACT
INTRODUCTION
Ct value). One Ct value corresponds to cycle of PCR amplification (ie, a two-fold difference in DNA content). 
2 P = .03). 
