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Multicenter Phase II and Translational Study of Cetuximab in Metastatic Colorectal Carcinoma Refractory to Irinotecan, Oxaliplatin, and Fluoropyrimidines

Heinz-Josef Lenz, Eric Van Cutsem, Shirin Khambata-Ford, Robert J. Mayer, Philip Gold, Philip Stella, Barry Mirtsching, Allen L. Cohn, Andrew W. Pippas, Nozar Azarnia, Zenta Tsuchihashi, David J. Mauro, Eric K. Rowinsky

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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
Cetuximab was administered as a 120-minute intravenous infusion at 400 mg/m² followed by weekly 60-minute infusions of 250 mg/m². Diphenhydramine, 50 mg intravenous, was administered before the first treatment and then before subsequent doses at the discretion of the investigator.

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
Histories, physical examinations, and safety assessments were performed pretreatment and weekly thereafter. Electrolytes, serum chemistries, liver and kidney function tests, clotting studies, urinalysis, and a chest radiograph, were monitored pretreatment, every 6 weeks, and at the end of treatment. To assess left ventricular function, multigated ventriculography or echocardiography was performed pretreatment, every 6 months and at the end of treatment.

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
Blood (5 mL) was sampled pretreatment, before the final cetuximab infusion during each course, and 4 weeks after completion of treatment. Serum was assayed for cetuximab using a Biacore (Uppsala, Sweden) assay method.³¹

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
Isolation of genomic DNA and descriptions of polymerase chain reaction (PCR) primers, PCR methodology, and analyses are found in online only Appendix 2.^32-36 The PCR assay measured the abundance of the EGFR gene relative to that of a control gene, VEST1.³⁷ Reactions were performed in triplicate using absolute quantification of Ct (threshold cycle) values. DiFi and A431 cell lines were used as positive controls. The difference in Ct values between the triplicate average for the EGFR and VEST1 probes was calculated ( Ct value). One Ct value corresponds to cycle of PCR amplification (ie, a two-fold difference in DNA content).

Statistical Analyses
The response rate was expected to range from 9% to 12% based on prior experience.^27,28 With a minimum enrollment of 250 patients, the 95% CI was estimated to be a maximum of 9%. All patients who received at least one dose of cetuximab were analyzed. All rates and proportions were compared using Fisher's exact test. The distribution of response duration, progression-free survival (PFS), and survival were estimated using the Kaplan-Meier product limit method.

Delta Ct values were related to best response using nonparametric analysis of variance. Proportional hazards (Cox) modeling was used to relate Ct values to PFS and overall survival.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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%).

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
Investigators determined that 43 (12.4%) patients had PRs, whereas the independent review committee determined that 40 patients (11.6%; 95% CI, 8.4% to 16.4%) responded, and 150 patients (43.4%) had either PRs or stable disease (Table 2). The median duration response was 4.2 months (95% CI, 4.1 to 4.4), and the median time to a response was 1.4 months (95% CI, 0.9 to 5.4). The median PFS was 1.4 months (95% CI, 1.4 to 2.1) and the 6-month PFS rate was 12.6% (95% CI, 8.9% to 16.3%). The median survival time was 6.6 months (95% CI, 5.6 to 7.6) and the 1-year survival rate was 27.4% (95% CI, 22.5% to 32.2%; Figs 1 and 2).

View larger version (7K): [in this window] [in a new window]   Fig 1. Kaplan-Meier curve for progression-free survival for all patients entered on study.

View larger version (7K): [in this window] [in a new window]   Fig 2. Kaplan-Meier curve for overall survival for all patients entered on study.

View larger version (10K): [in this window] [in a new window]   Fig 3. Kaplan-Meier curves for survival as a function of the intensity of cetuximab-related skin rash.

Pharmacokinetics and Immunokinetics
After multiple infusions, peak and trough serum cetuximab concentrations were relatively constant. Median (range) peak concentrations were 297 µg/mL (115 to 512 µg/mL), 298 µg/mL (122 to 500 µg/mL), and 279 µg/mL (179 to 598 µg/mL) after doses 1, 4, and 6, respectively, and median (range) trough concentrations were 0 µg/mL (0 µg/mL), 92 µg/mL (0 to 194 µg/mL), and 108 µg/mL (0 to 291 µg/mL). Interpatient variability was moderate, with coefficients of variability ranging from 31.6% to 59.7% for both peak and trough values.

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
Exons 18, 19, and 21 were fully sequenced in the CRC samples of all 39 patients from whom adequate tissue and informed consent could be obtained. With regard to response, 11, 19, and nine patients in this group experienced PRs, stable disease, and progressive disease, respectively. None of the EGFR TK domain mutations that had been associated with human malignancies and/or responsiveness to TK inhibitors, including codon 719 substitutions, exon 19 deletions, L858R, and L861Q, were detected. Three patients with stable disease appeared to have other mutations, including P753L and V689M (two samples), but these findings were not confirmed when the samples were reamplified, suggesting that the mutations were not in the original tumor.

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 Ct values, an index of EGFR gene copy number, to best response, PFS, and survival are shown in Figure 4 (A to C). No relationships between EGFR gene copy number and response or PFS were evident, but Ct values related to survival using Ct as a fixed covariate in a Cox model of survival (Wald ² P = .03).

View larger version (5K): [in this window] [in a new window]   Fig 4. Scatterplots showing distribution of delta Ct values, an index of EGFR gene copy number, as a function of (A) best response to cetuximab as assessed by an independent review committee; (B) progression-free survival; and (C) overall survival. Empty circles denote censored patients. No relationships between delta threshold cycle (Ct) values and both best objective response and progression-free survival were evident, but the relationship between delta Ct values and overall survival was significant using delta Ct as a fixed covariate in a Cox model of overall survival (Wald 2 P = .03).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

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.

	Appendix 1

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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.

	Appendix 2: Epidermal Growth Factor Receptor Sequencing and Amplifiation Studies—Methodology

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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 ( Ct value). One Ct value corresponds to cycle of PCR amplification (a two-fold difference in DNA content).

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

Dollar Amount Codes (A) <$10,000 (B) $10,000-99,999 (C) $100,000 (N/R) Not Required

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 

Conception and design: Heinz-Josef Lenz, Eric Van Cutsem, Shirin Khambata-Ford, Robert J. Mayer, Philip Gold, Philip Stella, Barry Mirtsching, Allen L. Cohn, Andrew W. Pippas, Nozar Azarnia, David J. Mauro Provision of study materials or patients: Heinz-Josef Lenz, Eric Van Cutsem, Shirin Khambata-Ford, Robert J. Mayer, Philip Gold, Philip Stella, Barry Mirtsching, Allen L. Cohn, Andrew W. Pippas Collection and assembly of data: Shirin Khambata-Ford, Nozar Azarnia, Zenta Tsuchihashi, David J. Mauro, Eric K. Rowinsky Data analysis and interpretation: Shirin Khambata-Ford, Nozar Azarnia, Zenta Tsuchihashi, David J. Mauro, Eric K. Rowinsky Manuscript writing: Shirin Khambata-Ford, Nozar Azarnia, Zenta Tsuchihashi, Eric K. Rowinsky Final approval of manuscript: Heinz-Josef Lenz, Eric Van Cutsem, Shirin Khambata-Ford, Robert J. Mayer, Philip Gold, Philip Stella, Barry Mirtsching, Allen L. Cohn, Andrew W. Pippas, Nozar Azarnia, Zenta Tsuchihashi, David J. Mauro, Eric K. Rowinsky

 

	ACKNOWLEDGMENTS

 
The authors are grateful to Christopher Harbison, William Geese, and John Feder for their efforts with mutation and gene copy number analyses.

	NOTES

 
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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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Appendix 1 Appendix 2: Epidermal Growth... Authors' Disclosures of... Author Contributions REFERENCES

 
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Submitted April 10, 2006; accepted July 7, 2006.

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