Cetuximab Shows Activity in Colorectal Cancer Patients With Tumors That Do Not Express the Epidermal Growth Factor Receptor by Immunohistoch
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《临床肿瘤学》
the Gastrointestinal Oncology Service and Departments of Medicine, Pathology, and Diagnostic Imaging, Memorial Sloan-Kettering Cancer Center, New York, NY
ABSTRACT
PURPOSE: To establish evidence of activity, or lack thereof, of cetuximab-based therapy in patients with refractory colorectal cancer with tumors that do not demonstrate epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC).
PATIENTS AND METHODS: Pharmacy computer records were reviewed to identify all patients who received cetuximab at Memorial Sloan-Kettering Cancer Center in a nonstudy setting during the first 3 months of cetuximab's commercial availability. Medical records of these patients were then reviewed to identify colorectal cancer patients who had experienced failure with a prior irinotecan-based regimen and who had a pathology report indicating an EGFR-negative tumor by IHC. Pathology slides from these patients were reviewed by a reference pathologist to confirm EGFR negativity, and computed tomography scans during cetuximab-based therapy were reviewed by a reference radiologist. Response rates were reported using WHO criteria.
RESULTS: Sixteen chemotherapy-refractory, EGFR-negative colorectal cancer patients who received cetuximab in a nonstudy setting were identified. Fourteen of these patients received cetuximab plus irinotecan, and two received cetuximab monotherapy. In the 16 patients, four major objective responses were seen (response rate, 25%; 95% CI, 4% to 46%).
CONCLUSION: Colorectal cancer patients with EGFR-negative tumors have the potential to respond to cetuximab-based therapies. EGFR analysis by current IHC techniques does not seem to have predictive value, and selection or exclusion of patients for cetuximab therapy on the basis of currently available EGFR IHC does not seem warranted.
INTRODUCTION
Cetuximab (C225, Erbitux; ImClone Systems Inc, New York, NY) is a chimeric immunoglobulin G1 monoclonal antibody that targets the extracellular domain of epidermal growth factor receptor (EGFR) with high specificity and affinity (0.1 to 0.2 nmol/L).1 It competitively inhibits endogenous ligand binding and thereby inhibits subsequent EGFR activation. Recent studies have demonstrated that cetuximab is active in metastatic colorectal cancer. Thus far, studies have been restricted to those patients whose tumors expressed EGFR by immunohistochemistry (IHC).
EGFR, also known as HER-1, is a transmembrane glycoprotein consisting of an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain.2,3 Binding of specific ligands, such as epidermal growth factor and transforming growth factor alpha, to the extracellular domain of the receptor results in the dimerization of the receptor either with another EGFR (homodimerization) or another member of the human EGFR family (heterodimerization). In turn, this stimulates phosphorylation of the intracellular tyrosine kinases on the receptor, which leads to the initiation of a cascade of intracellular signaling pathways that ultimately regulate cell proliferation, migration, adhesion, differentiation, and survival.4 After signal transduction, receptors are internalized and either downregulated or regenerated on the cell surface.5
Because of its high specificity for its known target, cetuximab has undergone a clinical development program grounded on the assumption that expression of EGFR, as measured by IHC, would be both quantitatively and qualitatively prognostic for cetuximab activity. On the basis of this assumption, clinical development of cetuximab has focused exclusively on tumor types with frequent high tumor expression of EGFR. Although tumor EGFR status can be quantified by several techniques, including IHC, enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, polymerase chain reaction, and fluorescence in situ hybridization, in practice, IHC has by far been the most commonly used method, with most clinical cetuximab trials reported to date requiring EGFR IHC positivity for study entry.
As a result of the development plan, experience with cetuximab in patients with tumors that are known not to express EGFR by IHC has been extremely limited. In abstract form, Lenz et al6 reported a small cohort of nine EGFR-negative patients who were entered onto a clinical trial of single-agent cetuximab. Two major objective responses were reported by the investigators. No other experience with cetuximab in EGFR-negative colorectal cancer patients has been reported to date.
In reviewing our data from EGFR-positive trials and the data of others, we noted that, within the population of patients with EGFR-positive tumors who have been treated with cetuximab, no relationship between intensity of EGFR expression and clinical activity has been demonstrated.7,8 This observation strongly challenges the hypothesis that the current IHC-determined EGFR-negative patients would be unable to respond to cetuximab. On this basis, a decision was made at the Memorial Sloan-Kettering Cancer Center (MSKCC) that EGFR-negative colorectal cancer patients would not be excluded from standard off-protocol treatment with cetuximab simply on the basis of EGFR status. It was left to physician discretion to determine whether to use cetuximab, and if so, whether to use it as a single agent or in conjunction with irinotecan. We have now reviewed our experience in EGFR-negative patients who were treated at MSKCC in the first 3 months of commercial availability of cetuximab. This article reports the outcome of that review.
PATIENTS AND METHODS
We used computerized pharmacy records to identify all patients at MSKCC who began a nonresearch treatment with cetuximab from February 2004, when the drug became commercially available, through the arbitrarily selected cutoff date of May 15, 2004 (to allow for adequate time to assess for clinical response). This project was approved by the Institutional Review Board/Privacy Board of MSKCC. We reviewed pathology records to identify IHC EGFR-negative patients with confirmed colorectal cancer, and we reviewed clinical records to identify and confirm clinical evidence of failure on prior irinotecan-based therapy. Sixteen patients meeting these criteria (criteria: received cetuximab, not on a research study, with colorectal cancer, with prior irinotecan treatment, and a negative IHC stain for EGFR) were identified, and all such patients are included in this analysis.
The IHC slides of all 16 patients used for EGFR determination were reviewed by a reference pathologist. The source of each specimen and the time interval from the acquisition of tissue (surgery or biopsy) to EGFR testing are documented (Table 1). The pathologist was aware that these 16 patients had been reported to be EGFR negative but was blinded to the clinical outcome of each patient with cetuximab.
Additional medical record information collected included carcinoembryonic antigen (CEA) levels before and after cetuximab treatment and documentation of the presence or absence of skin toxicities during cetuximab treatment, as available. Pretreatment and subsequent computed tomography or magnetic resonance imaging studies were reviewed, with the official reports obtained from the medical record, and used to provide the initial clinical assessment of response. Radiographic studies, including scans used to determine irinotecan resistance, were then reviewed by a reference radiologist blinded to the previously reported response data. Bidimensional evaluation of measurable lesions was reported using the WHO response criteria.9 Because this was a retrospective review with patients being treated by a number of different clinicians and not a prospective trial, the frequency at which scans were obtained varied widely, with the first follow-up scan obtained from 6 to 15 weeks after initiation of cetuximab therapy.
IHC
IHC stains for EGFR were performed as part of routine processing in the department of pathology of MSKCC using the standard streptavidin-biotin-peroxidase immunostaining procedure. The antibody used at our center is a mouse monoclonal anti-EGFR antibody (clone 31G7; Ventana Medical Systems, Inc, Tucson, AZ), which was used for all specimens. Both clone 31G7 and Dako (clone 2-18C9; Dako, Carpinteria, CA) antibodies are excellent for IHC studies, as demonstrated by their interchangeable use in IHC determinations in the published phase I and pharmacologic study of an EGFR antagonist.10 Although our institution, which deals with a large volume of specimens, does not use the Dako kit because of cost considerations, we retrospectively confirmed the concordance of the clone 31G7 antibody with the DakoCytomation EGFR kit (Dako; see Results). Appropriate positive and negative controls are used in each case.
The staining results are measured semiquantitatively on a scale of 0, 1+, 2+, and 3+. A stain was scored as follows: 0, there is no membranous staining in any of the tumor cells; 1+, there is membranous staining in less than 10% of the tumor cells with any intensity or in less than 30% of the tumor cells with weak intensity; 2+, there is staining in 10% to 30% of the tumor cells with moderate to strong intensity or staining in 30% to 50% of the tumor cells with weak to moderate intensity; and 3+, there is staining in more than 30% of the tumor cells with strong intensity or more than 50% of the tumor cells with any intensity. Representative examples of 0, 1+, 2+, and 3+ IHC staining for EGFR are demonstrated in Figure 1.
RESULTS
Patient Characteristics
Fifty-three patients treated with cetuximab were identified by computer pharmacy records; 16 (30%) of these patients were EGFR negative. These 16 patients included in this analysis ranged in age from 53 to 82 years old, with a median age of 69 years (Table 2). Eleven patients were male, and five were female. All patients had received prior therapy with multiple agents. All patients had experienced prior treatment with fluorouracil and with irinotecan for metastatic disease. Specifically, 15 of the 16 patients had experienced failure in an adequate therapeutic trial of an irinotecan-containing regimen for metastatic disease, with the remaining patient unable to tolerate irinotecan because of grade 4 diarrhea, which was recurrent on re-treatment with a lower dose (this patient was treated with cetuximab alone). Fifteen of 16 patients had also experienced clinical failure on regimens that had included oxaliplatin, with only one of the 16 patients not exposed to oxaliplatin. The median number of prior chemotherapy regimens for metastatic disease was two (range, two to five regimens).
Documentation in the clinical record of performance status on, or within 2 weeks before, the day of initiation of cetuximab was available in eight of 16 patients. The median Eastern Cooperative Oncology Group performance status in these patients was 2 (range, 1 to 2).
All patients received cetuximab on a once-a-week basis. Two patients were treated with cetuximab as a single agent, and 14 patients received cetuximab with irinotecan. Of these 14 patients, 12 received irinotecan on a weekly schedule, and two received it on an every other week schedule; none of these patients received irinotecan at a dose level higher than the level at which each patient had previously experienced failure.
Reference Pathologist Review
On thorough rereview of IHC slides from all 16 patients, 14 patients were confirmed as being EGFR negative. One patient who was initially reported as EGFR negative was determined on our rereview to be weakly EGFR positive (1/3+). One other patient, who was initially labeled EGFR negative, was reclassified as inassessable because, although the reviewed slide was EGFR negative, the slide was felt to contain largely adenoma and was considered to have inadequate invasive carcinoma volume to be considered sufficient for evaluation of tumor EGFR status. To avoid possible selection bias, these two patients are included in the analysis. As will be discussed later, neither patient experienced a clinical response to cetuximab.
Comparison of Anti-EGFR Clone 31G7 and Dako Antibodies
Additional tissue was available in 13 of our 16 patients to permit staining with the DakoCytomation EGFR kit. All 13 of these patients were scored as EGFR negative, with 11 of 13 patients demonstrating no staining at all and two patients (both nonresponders) showing trace focal cytoplasmic staining but no membranous staining. Therefore, retrospective concordance between the two antibodies was demonstrated.
Efficacy
All 16 patients who were initially screened as EGFR negative and who received any cetuximab during the designated time period were included in the efficacy analysis, although it is noteworthy that two patients received only one dose of cetuximab and one patient received only two doses of cetuximab before experiencing clinical deterioration secondary to disease-related complications and being removed from treatment. For the purposes of this analysis, these three patients are included in the denominator and classified as nonresponders.
Of the 16 patients, four (25%; 95% CI, 4% to 46%) achieved a partial response, with a greater than 50% reduction in the size of measurable lesions. Two additional patients had a minor response, with 39% and 32% reduction in the size of measurable lesions. All four partial response patients had at least one additional follow-up scan confirming the durability of response at 6 weeks or more (Fig 2). All four partial response and both minor response patients are still receiving treatment with irinotecan plus cetuximab at the time of this writing, with a median time of treatment thus far of 15 weeks (range, 8 to 17 weeks). Tumor responses are listed in Table 3. All percentage changes in tumor size are based on measurements by the reference radiologist, who was blinded to the reported measurements. There was complete concordance in terms of response status between the reports in the medical record and the response assessments of the reference radiologist.
Overall, of the 16 EGFR-negative patients who were treated, seven achieved some degree of tumor control with cetuximab-based therapy. One additional patient was treated for 15 weeks with a greater than 50% drop in CEA (from 832 to 374 ng/mL), which was followed by a subsequent increase in CEA to 613 ng/mL. No scan was obtained at the time of best CEA response, and the patient is classified for this analysis as having progression of disease as best response, despite 15 weeks on therapy.
Our data set is too small to evaluate for a clear correlation between rash and response; however, there was clearly no strong correlation in this area. Of four patients with a partial response, two had no report of skin rash, and two were reported to have developed a mild (grade 1) rash. Both patients who achieved a minor response had documentation of a rash (one with grade 1 and the other with grade 2 rash), which did not require discontinuation of therapy. The one patient with stable disease developed a grade 1 rash. Of the five patients with progression of disease, two did not develop a rash, whereas three patients developed a grade 1 rash.
All six of the patients with tumor shrinkage had received cetuximab plus irinotecan. Of the two patients who received single-agent cetuximab, one achieved stable disease (ongoing at 10 weeks), and one suffered clinical deterioration after two doses and was removed from treatment.
DISCUSSION
Currently, cetuximab is approved for use by the US Food and Drug Administration in the treatment of colorectal cancer. Specifically, it is approved for use in conjunction with irinotecan (Camptosar; Pfizer, New York, NY) in irinotecan-refractory colorectal cancer patients or as a single agent in patients with a contraindication to irinotecan. More specifically, presumably given the fact that the clinical trials leading to this approval excluded patients who lacked EGFR expression by IHC, the approval is currently restricted to those patients whose tumors express EGFR. By IHC, approximately 70% to 75% of human colorectal carcinomas express EGFR.
Our experience in the first 3 months of commercial cetuximab availability confirms our hypothesis that irinotecan-refractory, EGFR-negative colorectal cancer patients, as defined by routine IHC analysis, can respond to cetuximab-based therapy. The 25% response rate to cetuximab plus irinotecan therapy demonstrated in our EGFR-negative patient series is the first response rate for the use of this combination therapy reported to our knowledge in this population and is indistinguishable from the 23% response rate seen in two separate cetuximab-plus-irinotecan clinical trials in EGFR-positive patients.11,8 Although the 95% CIs are wide, the concept that cetuximab is inactive in EGFR-negative colorectal cancer is strongly refuted by our data and by the cetuximab trials that have failed to demonstrate a relationship between response and EGFR expression levels. Similarly, the 22% response rate (two of nine patients) demonstrated by Lenz et al6 in a small cohort of EGFR-negative colorectal patients receiving single-agent cetuximab is unlikely to be substantially inferior to the 9% to 12% response rates reported in single-agent cetuximab trials in EGFR-positive patients.7,8
Our methodology is designed to effectively address the major concerns typically associated with retrospective analyses. By using computerized pharmacy records to identify all patients treated with cetuximab at our institution within the specified time frame and then reviewing pathology records to assess documentation of EGFR positivity or negativity, we have effectively excluded the risk of recall bias from our analysis. The potential for selection bias is also effectively addressed by inclusion of all patients who met initially described eligibility criteria within the specified time frame, regardless of whether they received a meaningful course of therapy or just one or two treatments and regardless of whether or not their EGFR-negative status was confirmed. Assessment bias was addressed by use of a reference radiologist who was blinded to reported clinical and radiologic data and who independently performed determinations of response or failure on all patients. All patients were heavily pretreated and had a median performance status that was certainly not better than that of patients enrolled onto clinical trials. Hence, the conclusions drawn from this retrospective series are clinically relevant and not simply attributable to a beta error.
Despite the fact that clinical trials have been largely limited to patients who had tumors that were EGFR positive by IHC, considerable evidence exists to suggest that EGFR positivity, at least as measured by this method, has little or no predictive value in terms of cetuximab's activity in colorectal cancer.7,8 In the reported trials of EGFR-positive colorectal cancer, response rates for 1+, 2+, and 3+ tumors were essentially the same, with no tendency towards a higher response rate with higher EGFR expression.7,11,8
The obvious question engendered by our finding is why does a specific monoclonal antibody to the extracellular domain of EGFR work in presumably EGFR-negative patients One potential explanation is simply inconsistent methodology and interpretation of EGFR IHC expression in tumor samples. Currently, there are potential vagaries in the scoring system established for the interpretation of EGFR expression, with some subjectivity and interobserver variability in the definitions of absent, low, intermediate, and high expression. The fact that rereview by a reference pathologist reclassified two of 16 presumably EGFR-negative patients at a major teaching hospital illustrates the imperfections in current IHC methodology for both the performance and the interpretation of EGFR.
Several studies have reported variability in EGFR staining depending on the tissue fixation technique used, possibly leading to falsely negative samples.12-14 Also, most EGFR analysis has been carried out on archived primary tumor specimens that may have been kept for months to years before EGFR analysis. A recent prospective study evaluated EGFR expression by IHC in paraffin-embedded tumor tissue from 40 patients (10 with non–small-cell cancer, 10 with squamous cell carcinoma of the head and neck, and 20 with colorectal adenocarcinoma); each tissue sample was stained with eight commonly used preservation reagents and prospectively evaluated for EGFR expression at six time points extending to 24 months.15 Significant variability in EGFR immunoreactivity was seen depending on fixation methodology. The most striking finding from this study, however, was the dramatic decline in EGFR staining intensity with increasing storage time of the tissue samples, leading to many more EGFR-negative or EGFR 0 scorings in older specimens. Because EGFR evaluation in practice, as in the cetuximab registration trials, has been based on archived tissues from numerous sources and therapeutic time points, the fixation reagents and methodology and age of the specimens have invariably been unknown. Therefore, the current EGFR IHC methodology is subject to many variables and confounding factors that may contribute to the fact that EFGR by IHC is, thus far, an inadequate predictive marker for response.
An additional concern is that metastatic tumor specimens are often biologically distinct from the primary tumors, perhaps reflecting the acquisition of additional mutations conferring growth advantage and the cumulative effect of clonal selection in the setting of exposure to multiple prior chemotherapeutic agents. Highlighting the potential impact of these biologic differences, some solid tumor studies have identified statistically significant differences in IHC EGFR expression between an individual patient's primary tumor and metastatic tissue specimen.16 Because of tissue availability, the primary tumor is frequently used to establish the patient's EGFR status, but it is the metastases that are being treated with cetuximab.
An alternative explanation for the cetuximab-based responses in EGFR-negative patients delves deeper into the biology of EGFR. The human epidermoid carcinoma cell line A431, which is by far the most commonly used line for the study of EGFR structure and function because of its unusually high numbers of EGFR receptors (2 to 3 x 106/cell), is the source from which most commercially available anti-EGFR antibodies for IHC are derived, including the DakoCytomation EGFR kit.17 Two distinct EGFRs have been identified in A431 cells by epidermal growth factor–binding studies; these are a major class of low-affinity EGFR (representing approximately 95% of the receptors) and a minor class of high-affinity EGFR (representing approximately 5% of the receptors), with binding affinities differing by an order of magnitude.18-20 Data from multiple investigations suggest that the high-affinity EGFRs are the predominant biologically active receptors that lead to protein tyrosine kinase activation, whereas the low-affinity EGFRs do not contribute significantly to signal transduction.20-23 The current EGFR IHC detection systems used today that use anti-EGFR antibodies derived from A431 cells do not distinguish between these two distinct EGFRs. In fact, there are no published studies that have investigated the presence and distribution of high- and low-affinity EGFR in colorectal cancers. One could speculate that patients with few overall EGFRs below the threshold of IHC detection (10 to 1,000 receptors/cell) but with a high ratio of high-affinity to low-affinity EGFRs could be responsive to an EGFR-targeted agent.
Aside from these potential inaccuracies in IHC determination of relevant membrane levels of EGFR, another potential explanation for the lack of correlation between EGFR expression and antitumor activity may lie in the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Similar to other clinically effective chimeric monoclonal antibodies such as rituximab, cetuximab may be instigating an ADCC reaction, leading to indirect antitumor activity by the recruitment of cytotoxic host effector cells such as monocytes and natural-killer cells. The possibility of such an ADCC reaction was suggested by a study revealing reduced in vivo anti-A431 xenograft tumor activity using 225F(ab')2, which lacks the Fc portion of the monoclonal antibody, compared with the native 225 monoclonal antibody, despite equivalent pharmacologic EGFR blockade.24 In addition, a recent study of a humanized anti-EGFR antibody and a rat anti-EGFR antibody demonstrated that both generated a potent, predominantly natural-killer cell–mediated ADCC when applied against monolayers of an established squamous cell carcinoma of the head and neck cell line.25 Hence, this indirect antitumor mechanism also deserves further exploration.
Our small data set does not accurately establish the exact degree of activity for cetuximab-based therapy in EGFR-negative, chemotherapy-refractory colorectal cancer; it also does not tell us definitively whether the activity level is the same, better, or worse for EGFR-negative patients compared with EGFR-positive patients. It should be noted that the size of an adequately powered, randomized, equivalence trial to assess whether or not the activity levels are the same in IHC-determined EGFR-positive and EGFR-negative patients would be prohibitive, and such a trial will never be performed. Prospective trials to assess the activity level of cetuximab, both alone and in combination with irinotecan, are planned in EGFR-negative patients, but these trials will necessarily be single-arm, uncontrolled trials, and the comparisons of results with the results of EGFR-positive patient trials will necessarily be only exploratory in nature.
The finding of activity with cetuximab-based therapy in EGFR-negative patients has a number of important implications. Most importantly, it suggests that the current routine practice of tumor IHC EGFR testing for the purpose of selecting cetuximab therapy is inappropriate because patients who could potentially benefit from cetuximab may be excluded from treatment. This has important ramifications for some third-party payors who have adopted the position of restricting reimbursement for cetuximab to patients with EGFR-positive colorectal tumors. Such reimbursement restrictions do not seem to be clinically justified. Just as importantly, the use of cetuximab in a noncolorectal tumor simply because the tumor is strongly EGFR positive is equally unfounded.
It may well be that a presently undetermined IHC assay, possibly for high-affinity receptors, or another quantitative or qualitative assay for EGFR, such as reverse transcription polymerase chain reaction, will predict for cetuximab activity. However, to date, such a marker has yet to be identified and validated.
Authors' Disclosures of Potential Conflicts of Interest
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. Consultant/Advisory Role: Nancy Kemeny, Pfizer; Lawrence Schwartz, Imclone Systems; David P. Kelsen, Pfizer; Leonard B. Saltz, Genentech, Pfizer, Roche, Sanofi. Honoraria: Nancy Kemeny, Pfizer; David P. Kelsen, Pfizer; Leonard B. Saltz, Bristol-Myers Squibb. Research Funding: Nancy Kemeny, Pfizer; Leonard B. Saltz, Bristol-Myers Squibb, Pfizer, Imclone. 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 form and the Disclosures of Potential Conflicts of Interest section of Information for Contributors found in the front of every issue.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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ABSTRACT
PURPOSE: To establish evidence of activity, or lack thereof, of cetuximab-based therapy in patients with refractory colorectal cancer with tumors that do not demonstrate epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC).
PATIENTS AND METHODS: Pharmacy computer records were reviewed to identify all patients who received cetuximab at Memorial Sloan-Kettering Cancer Center in a nonstudy setting during the first 3 months of cetuximab's commercial availability. Medical records of these patients were then reviewed to identify colorectal cancer patients who had experienced failure with a prior irinotecan-based regimen and who had a pathology report indicating an EGFR-negative tumor by IHC. Pathology slides from these patients were reviewed by a reference pathologist to confirm EGFR negativity, and computed tomography scans during cetuximab-based therapy were reviewed by a reference radiologist. Response rates were reported using WHO criteria.
RESULTS: Sixteen chemotherapy-refractory, EGFR-negative colorectal cancer patients who received cetuximab in a nonstudy setting were identified. Fourteen of these patients received cetuximab plus irinotecan, and two received cetuximab monotherapy. In the 16 patients, four major objective responses were seen (response rate, 25%; 95% CI, 4% to 46%).
CONCLUSION: Colorectal cancer patients with EGFR-negative tumors have the potential to respond to cetuximab-based therapies. EGFR analysis by current IHC techniques does not seem to have predictive value, and selection or exclusion of patients for cetuximab therapy on the basis of currently available EGFR IHC does not seem warranted.
INTRODUCTION
Cetuximab (C225, Erbitux; ImClone Systems Inc, New York, NY) is a chimeric immunoglobulin G1 monoclonal antibody that targets the extracellular domain of epidermal growth factor receptor (EGFR) with high specificity and affinity (0.1 to 0.2 nmol/L).1 It competitively inhibits endogenous ligand binding and thereby inhibits subsequent EGFR activation. Recent studies have demonstrated that cetuximab is active in metastatic colorectal cancer. Thus far, studies have been restricted to those patients whose tumors expressed EGFR by immunohistochemistry (IHC).
EGFR, also known as HER-1, is a transmembrane glycoprotein consisting of an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain.2,3 Binding of specific ligands, such as epidermal growth factor and transforming growth factor alpha, to the extracellular domain of the receptor results in the dimerization of the receptor either with another EGFR (homodimerization) or another member of the human EGFR family (heterodimerization). In turn, this stimulates phosphorylation of the intracellular tyrosine kinases on the receptor, which leads to the initiation of a cascade of intracellular signaling pathways that ultimately regulate cell proliferation, migration, adhesion, differentiation, and survival.4 After signal transduction, receptors are internalized and either downregulated or regenerated on the cell surface.5
Because of its high specificity for its known target, cetuximab has undergone a clinical development program grounded on the assumption that expression of EGFR, as measured by IHC, would be both quantitatively and qualitatively prognostic for cetuximab activity. On the basis of this assumption, clinical development of cetuximab has focused exclusively on tumor types with frequent high tumor expression of EGFR. Although tumor EGFR status can be quantified by several techniques, including IHC, enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, polymerase chain reaction, and fluorescence in situ hybridization, in practice, IHC has by far been the most commonly used method, with most clinical cetuximab trials reported to date requiring EGFR IHC positivity for study entry.
As a result of the development plan, experience with cetuximab in patients with tumors that are known not to express EGFR by IHC has been extremely limited. In abstract form, Lenz et al6 reported a small cohort of nine EGFR-negative patients who were entered onto a clinical trial of single-agent cetuximab. Two major objective responses were reported by the investigators. No other experience with cetuximab in EGFR-negative colorectal cancer patients has been reported to date.
In reviewing our data from EGFR-positive trials and the data of others, we noted that, within the population of patients with EGFR-positive tumors who have been treated with cetuximab, no relationship between intensity of EGFR expression and clinical activity has been demonstrated.7,8 This observation strongly challenges the hypothesis that the current IHC-determined EGFR-negative patients would be unable to respond to cetuximab. On this basis, a decision was made at the Memorial Sloan-Kettering Cancer Center (MSKCC) that EGFR-negative colorectal cancer patients would not be excluded from standard off-protocol treatment with cetuximab simply on the basis of EGFR status. It was left to physician discretion to determine whether to use cetuximab, and if so, whether to use it as a single agent or in conjunction with irinotecan. We have now reviewed our experience in EGFR-negative patients who were treated at MSKCC in the first 3 months of commercial availability of cetuximab. This article reports the outcome of that review.
PATIENTS AND METHODS
We used computerized pharmacy records to identify all patients at MSKCC who began a nonresearch treatment with cetuximab from February 2004, when the drug became commercially available, through the arbitrarily selected cutoff date of May 15, 2004 (to allow for adequate time to assess for clinical response). This project was approved by the Institutional Review Board/Privacy Board of MSKCC. We reviewed pathology records to identify IHC EGFR-negative patients with confirmed colorectal cancer, and we reviewed clinical records to identify and confirm clinical evidence of failure on prior irinotecan-based therapy. Sixteen patients meeting these criteria (criteria: received cetuximab, not on a research study, with colorectal cancer, with prior irinotecan treatment, and a negative IHC stain for EGFR) were identified, and all such patients are included in this analysis.
The IHC slides of all 16 patients used for EGFR determination were reviewed by a reference pathologist. The source of each specimen and the time interval from the acquisition of tissue (surgery or biopsy) to EGFR testing are documented (Table 1). The pathologist was aware that these 16 patients had been reported to be EGFR negative but was blinded to the clinical outcome of each patient with cetuximab.
Additional medical record information collected included carcinoembryonic antigen (CEA) levels before and after cetuximab treatment and documentation of the presence or absence of skin toxicities during cetuximab treatment, as available. Pretreatment and subsequent computed tomography or magnetic resonance imaging studies were reviewed, with the official reports obtained from the medical record, and used to provide the initial clinical assessment of response. Radiographic studies, including scans used to determine irinotecan resistance, were then reviewed by a reference radiologist blinded to the previously reported response data. Bidimensional evaluation of measurable lesions was reported using the WHO response criteria.9 Because this was a retrospective review with patients being treated by a number of different clinicians and not a prospective trial, the frequency at which scans were obtained varied widely, with the first follow-up scan obtained from 6 to 15 weeks after initiation of cetuximab therapy.
IHC
IHC stains for EGFR were performed as part of routine processing in the department of pathology of MSKCC using the standard streptavidin-biotin-peroxidase immunostaining procedure. The antibody used at our center is a mouse monoclonal anti-EGFR antibody (clone 31G7; Ventana Medical Systems, Inc, Tucson, AZ), which was used for all specimens. Both clone 31G7 and Dako (clone 2-18C9; Dako, Carpinteria, CA) antibodies are excellent for IHC studies, as demonstrated by their interchangeable use in IHC determinations in the published phase I and pharmacologic study of an EGFR antagonist.10 Although our institution, which deals with a large volume of specimens, does not use the Dako kit because of cost considerations, we retrospectively confirmed the concordance of the clone 31G7 antibody with the DakoCytomation EGFR kit (Dako; see Results). Appropriate positive and negative controls are used in each case.
The staining results are measured semiquantitatively on a scale of 0, 1+, 2+, and 3+. A stain was scored as follows: 0, there is no membranous staining in any of the tumor cells; 1+, there is membranous staining in less than 10% of the tumor cells with any intensity or in less than 30% of the tumor cells with weak intensity; 2+, there is staining in 10% to 30% of the tumor cells with moderate to strong intensity or staining in 30% to 50% of the tumor cells with weak to moderate intensity; and 3+, there is staining in more than 30% of the tumor cells with strong intensity or more than 50% of the tumor cells with any intensity. Representative examples of 0, 1+, 2+, and 3+ IHC staining for EGFR are demonstrated in Figure 1.
RESULTS
Patient Characteristics
Fifty-three patients treated with cetuximab were identified by computer pharmacy records; 16 (30%) of these patients were EGFR negative. These 16 patients included in this analysis ranged in age from 53 to 82 years old, with a median age of 69 years (Table 2). Eleven patients were male, and five were female. All patients had received prior therapy with multiple agents. All patients had experienced prior treatment with fluorouracil and with irinotecan for metastatic disease. Specifically, 15 of the 16 patients had experienced failure in an adequate therapeutic trial of an irinotecan-containing regimen for metastatic disease, with the remaining patient unable to tolerate irinotecan because of grade 4 diarrhea, which was recurrent on re-treatment with a lower dose (this patient was treated with cetuximab alone). Fifteen of 16 patients had also experienced clinical failure on regimens that had included oxaliplatin, with only one of the 16 patients not exposed to oxaliplatin. The median number of prior chemotherapy regimens for metastatic disease was two (range, two to five regimens).
Documentation in the clinical record of performance status on, or within 2 weeks before, the day of initiation of cetuximab was available in eight of 16 patients. The median Eastern Cooperative Oncology Group performance status in these patients was 2 (range, 1 to 2).
All patients received cetuximab on a once-a-week basis. Two patients were treated with cetuximab as a single agent, and 14 patients received cetuximab with irinotecan. Of these 14 patients, 12 received irinotecan on a weekly schedule, and two received it on an every other week schedule; none of these patients received irinotecan at a dose level higher than the level at which each patient had previously experienced failure.
Reference Pathologist Review
On thorough rereview of IHC slides from all 16 patients, 14 patients were confirmed as being EGFR negative. One patient who was initially reported as EGFR negative was determined on our rereview to be weakly EGFR positive (1/3+). One other patient, who was initially labeled EGFR negative, was reclassified as inassessable because, although the reviewed slide was EGFR negative, the slide was felt to contain largely adenoma and was considered to have inadequate invasive carcinoma volume to be considered sufficient for evaluation of tumor EGFR status. To avoid possible selection bias, these two patients are included in the analysis. As will be discussed later, neither patient experienced a clinical response to cetuximab.
Comparison of Anti-EGFR Clone 31G7 and Dako Antibodies
Additional tissue was available in 13 of our 16 patients to permit staining with the DakoCytomation EGFR kit. All 13 of these patients were scored as EGFR negative, with 11 of 13 patients demonstrating no staining at all and two patients (both nonresponders) showing trace focal cytoplasmic staining but no membranous staining. Therefore, retrospective concordance between the two antibodies was demonstrated.
Efficacy
All 16 patients who were initially screened as EGFR negative and who received any cetuximab during the designated time period were included in the efficacy analysis, although it is noteworthy that two patients received only one dose of cetuximab and one patient received only two doses of cetuximab before experiencing clinical deterioration secondary to disease-related complications and being removed from treatment. For the purposes of this analysis, these three patients are included in the denominator and classified as nonresponders.
Of the 16 patients, four (25%; 95% CI, 4% to 46%) achieved a partial response, with a greater than 50% reduction in the size of measurable lesions. Two additional patients had a minor response, with 39% and 32% reduction in the size of measurable lesions. All four partial response patients had at least one additional follow-up scan confirming the durability of response at 6 weeks or more (Fig 2). All four partial response and both minor response patients are still receiving treatment with irinotecan plus cetuximab at the time of this writing, with a median time of treatment thus far of 15 weeks (range, 8 to 17 weeks). Tumor responses are listed in Table 3. All percentage changes in tumor size are based on measurements by the reference radiologist, who was blinded to the reported measurements. There was complete concordance in terms of response status between the reports in the medical record and the response assessments of the reference radiologist.
Overall, of the 16 EGFR-negative patients who were treated, seven achieved some degree of tumor control with cetuximab-based therapy. One additional patient was treated for 15 weeks with a greater than 50% drop in CEA (from 832 to 374 ng/mL), which was followed by a subsequent increase in CEA to 613 ng/mL. No scan was obtained at the time of best CEA response, and the patient is classified for this analysis as having progression of disease as best response, despite 15 weeks on therapy.
Our data set is too small to evaluate for a clear correlation between rash and response; however, there was clearly no strong correlation in this area. Of four patients with a partial response, two had no report of skin rash, and two were reported to have developed a mild (grade 1) rash. Both patients who achieved a minor response had documentation of a rash (one with grade 1 and the other with grade 2 rash), which did not require discontinuation of therapy. The one patient with stable disease developed a grade 1 rash. Of the five patients with progression of disease, two did not develop a rash, whereas three patients developed a grade 1 rash.
All six of the patients with tumor shrinkage had received cetuximab plus irinotecan. Of the two patients who received single-agent cetuximab, one achieved stable disease (ongoing at 10 weeks), and one suffered clinical deterioration after two doses and was removed from treatment.
DISCUSSION
Currently, cetuximab is approved for use by the US Food and Drug Administration in the treatment of colorectal cancer. Specifically, it is approved for use in conjunction with irinotecan (Camptosar; Pfizer, New York, NY) in irinotecan-refractory colorectal cancer patients or as a single agent in patients with a contraindication to irinotecan. More specifically, presumably given the fact that the clinical trials leading to this approval excluded patients who lacked EGFR expression by IHC, the approval is currently restricted to those patients whose tumors express EGFR. By IHC, approximately 70% to 75% of human colorectal carcinomas express EGFR.
Our experience in the first 3 months of commercial cetuximab availability confirms our hypothesis that irinotecan-refractory, EGFR-negative colorectal cancer patients, as defined by routine IHC analysis, can respond to cetuximab-based therapy. The 25% response rate to cetuximab plus irinotecan therapy demonstrated in our EGFR-negative patient series is the first response rate for the use of this combination therapy reported to our knowledge in this population and is indistinguishable from the 23% response rate seen in two separate cetuximab-plus-irinotecan clinical trials in EGFR-positive patients.11,8 Although the 95% CIs are wide, the concept that cetuximab is inactive in EGFR-negative colorectal cancer is strongly refuted by our data and by the cetuximab trials that have failed to demonstrate a relationship between response and EGFR expression levels. Similarly, the 22% response rate (two of nine patients) demonstrated by Lenz et al6 in a small cohort of EGFR-negative colorectal patients receiving single-agent cetuximab is unlikely to be substantially inferior to the 9% to 12% response rates reported in single-agent cetuximab trials in EGFR-positive patients.7,8
Our methodology is designed to effectively address the major concerns typically associated with retrospective analyses. By using computerized pharmacy records to identify all patients treated with cetuximab at our institution within the specified time frame and then reviewing pathology records to assess documentation of EGFR positivity or negativity, we have effectively excluded the risk of recall bias from our analysis. The potential for selection bias is also effectively addressed by inclusion of all patients who met initially described eligibility criteria within the specified time frame, regardless of whether they received a meaningful course of therapy or just one or two treatments and regardless of whether or not their EGFR-negative status was confirmed. Assessment bias was addressed by use of a reference radiologist who was blinded to reported clinical and radiologic data and who independently performed determinations of response or failure on all patients. All patients were heavily pretreated and had a median performance status that was certainly not better than that of patients enrolled onto clinical trials. Hence, the conclusions drawn from this retrospective series are clinically relevant and not simply attributable to a beta error.
Despite the fact that clinical trials have been largely limited to patients who had tumors that were EGFR positive by IHC, considerable evidence exists to suggest that EGFR positivity, at least as measured by this method, has little or no predictive value in terms of cetuximab's activity in colorectal cancer.7,8 In the reported trials of EGFR-positive colorectal cancer, response rates for 1+, 2+, and 3+ tumors were essentially the same, with no tendency towards a higher response rate with higher EGFR expression.7,11,8
The obvious question engendered by our finding is why does a specific monoclonal antibody to the extracellular domain of EGFR work in presumably EGFR-negative patients One potential explanation is simply inconsistent methodology and interpretation of EGFR IHC expression in tumor samples. Currently, there are potential vagaries in the scoring system established for the interpretation of EGFR expression, with some subjectivity and interobserver variability in the definitions of absent, low, intermediate, and high expression. The fact that rereview by a reference pathologist reclassified two of 16 presumably EGFR-negative patients at a major teaching hospital illustrates the imperfections in current IHC methodology for both the performance and the interpretation of EGFR.
Several studies have reported variability in EGFR staining depending on the tissue fixation technique used, possibly leading to falsely negative samples.12-14 Also, most EGFR analysis has been carried out on archived primary tumor specimens that may have been kept for months to years before EGFR analysis. A recent prospective study evaluated EGFR expression by IHC in paraffin-embedded tumor tissue from 40 patients (10 with non–small-cell cancer, 10 with squamous cell carcinoma of the head and neck, and 20 with colorectal adenocarcinoma); each tissue sample was stained with eight commonly used preservation reagents and prospectively evaluated for EGFR expression at six time points extending to 24 months.15 Significant variability in EGFR immunoreactivity was seen depending on fixation methodology. The most striking finding from this study, however, was the dramatic decline in EGFR staining intensity with increasing storage time of the tissue samples, leading to many more EGFR-negative or EGFR 0 scorings in older specimens. Because EGFR evaluation in practice, as in the cetuximab registration trials, has been based on archived tissues from numerous sources and therapeutic time points, the fixation reagents and methodology and age of the specimens have invariably been unknown. Therefore, the current EGFR IHC methodology is subject to many variables and confounding factors that may contribute to the fact that EFGR by IHC is, thus far, an inadequate predictive marker for response.
An additional concern is that metastatic tumor specimens are often biologically distinct from the primary tumors, perhaps reflecting the acquisition of additional mutations conferring growth advantage and the cumulative effect of clonal selection in the setting of exposure to multiple prior chemotherapeutic agents. Highlighting the potential impact of these biologic differences, some solid tumor studies have identified statistically significant differences in IHC EGFR expression between an individual patient's primary tumor and metastatic tissue specimen.16 Because of tissue availability, the primary tumor is frequently used to establish the patient's EGFR status, but it is the metastases that are being treated with cetuximab.
An alternative explanation for the cetuximab-based responses in EGFR-negative patients delves deeper into the biology of EGFR. The human epidermoid carcinoma cell line A431, which is by far the most commonly used line for the study of EGFR structure and function because of its unusually high numbers of EGFR receptors (2 to 3 x 106/cell), is the source from which most commercially available anti-EGFR antibodies for IHC are derived, including the DakoCytomation EGFR kit.17 Two distinct EGFRs have been identified in A431 cells by epidermal growth factor–binding studies; these are a major class of low-affinity EGFR (representing approximately 95% of the receptors) and a minor class of high-affinity EGFR (representing approximately 5% of the receptors), with binding affinities differing by an order of magnitude.18-20 Data from multiple investigations suggest that the high-affinity EGFRs are the predominant biologically active receptors that lead to protein tyrosine kinase activation, whereas the low-affinity EGFRs do not contribute significantly to signal transduction.20-23 The current EGFR IHC detection systems used today that use anti-EGFR antibodies derived from A431 cells do not distinguish between these two distinct EGFRs. In fact, there are no published studies that have investigated the presence and distribution of high- and low-affinity EGFR in colorectal cancers. One could speculate that patients with few overall EGFRs below the threshold of IHC detection (10 to 1,000 receptors/cell) but with a high ratio of high-affinity to low-affinity EGFRs could be responsive to an EGFR-targeted agent.
Aside from these potential inaccuracies in IHC determination of relevant membrane levels of EGFR, another potential explanation for the lack of correlation between EGFR expression and antitumor activity may lie in the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Similar to other clinically effective chimeric monoclonal antibodies such as rituximab, cetuximab may be instigating an ADCC reaction, leading to indirect antitumor activity by the recruitment of cytotoxic host effector cells such as monocytes and natural-killer cells. The possibility of such an ADCC reaction was suggested by a study revealing reduced in vivo anti-A431 xenograft tumor activity using 225F(ab')2, which lacks the Fc portion of the monoclonal antibody, compared with the native 225 monoclonal antibody, despite equivalent pharmacologic EGFR blockade.24 In addition, a recent study of a humanized anti-EGFR antibody and a rat anti-EGFR antibody demonstrated that both generated a potent, predominantly natural-killer cell–mediated ADCC when applied against monolayers of an established squamous cell carcinoma of the head and neck cell line.25 Hence, this indirect antitumor mechanism also deserves further exploration.
Our small data set does not accurately establish the exact degree of activity for cetuximab-based therapy in EGFR-negative, chemotherapy-refractory colorectal cancer; it also does not tell us definitively whether the activity level is the same, better, or worse for EGFR-negative patients compared with EGFR-positive patients. It should be noted that the size of an adequately powered, randomized, equivalence trial to assess whether or not the activity levels are the same in IHC-determined EGFR-positive and EGFR-negative patients would be prohibitive, and such a trial will never be performed. Prospective trials to assess the activity level of cetuximab, both alone and in combination with irinotecan, are planned in EGFR-negative patients, but these trials will necessarily be single-arm, uncontrolled trials, and the comparisons of results with the results of EGFR-positive patient trials will necessarily be only exploratory in nature.
The finding of activity with cetuximab-based therapy in EGFR-negative patients has a number of important implications. Most importantly, it suggests that the current routine practice of tumor IHC EGFR testing for the purpose of selecting cetuximab therapy is inappropriate because patients who could potentially benefit from cetuximab may be excluded from treatment. This has important ramifications for some third-party payors who have adopted the position of restricting reimbursement for cetuximab to patients with EGFR-positive colorectal tumors. Such reimbursement restrictions do not seem to be clinically justified. Just as importantly, the use of cetuximab in a noncolorectal tumor simply because the tumor is strongly EGFR positive is equally unfounded.
It may well be that a presently undetermined IHC assay, possibly for high-affinity receptors, or another quantitative or qualitative assay for EGFR, such as reverse transcription polymerase chain reaction, will predict for cetuximab activity. However, to date, such a marker has yet to be identified and validated.
Authors' Disclosures of Potential Conflicts of Interest
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. Consultant/Advisory Role: Nancy Kemeny, Pfizer; Lawrence Schwartz, Imclone Systems; David P. Kelsen, Pfizer; Leonard B. Saltz, Genentech, Pfizer, Roche, Sanofi. Honoraria: Nancy Kemeny, Pfizer; David P. Kelsen, Pfizer; Leonard B. Saltz, Bristol-Myers Squibb. Research Funding: Nancy Kemeny, Pfizer; Leonard B. Saltz, Bristol-Myers Squibb, Pfizer, Imclone. 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 form and the Disclosures of Potential Conflicts of Interest section of Information for Contributors found in the front of every issue.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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