Clinical Use of Monoclonal Antibodies to the Epidermal Growth Factor Receptor in Colorectal Cancer

Monoclonal antibodies to the epidermal growth factor receptor (EGFR) are among the promising novel targeted therapies being explored in colorectal cancer. Two such agents that inhibit EGFR signaling by interfering with ligand-binding are cetuximab (Erbitux) and panitumumab (Vectibix). This review will address the use of cetuximab and panitumumab in chemotherapy-refractory colorectal cancer as well as in front-line therapy for the disease, consider predictors of response and resistance, and outline comparisons between these agents.

Colorectal cancer remains a common disease in the United States. The projected incidence for 2007 is 153,760 cases, and 52,180 deaths are expected.[1] Advances were made in the systemic therapy of metastatic colorectal cancer after the introduction of the novel cytotoxics irinotecan (Camptosar) and oxaliplatin (Eloxatin), extending median survival for metastatic disease from 9 months with fluorouracil (5-FU) and leucovorin, to longer than 17 months with first-line use of oxaliplatin together with 5-FU, and longer than 20 months for patients who are well enough to receive, and who have access to, all three drugs at some time in their treatment course.[2-4]

Novel targets are also being explored. A clear survival advantage has been demonstrated for the addition of bevacizumab (Avastin), a monoclonal antibody that targets the vascular endothelial growth factor (VEGF).[5]* Also active, but less well-studied in front-line therapy, are agents that target the epidermal growth factor receptor (EGFR). Such drugs are the focus of this review.

Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR or HER1) is a 170-kd polypeptide tyrosine kinase growth factor receptor.[6] Along with HER2, HER3, and HER4, it is a member of the HER receptor family and an important mediator of cell proliferation, differentiation, and survival. The extracellular region of the EGFR contains both a ligand-binding domain and a dimerization loop; the tyrosine kinase domain is located in the molecule's cytoplasmic region. A putative nuclear localization sequence has been identified in the juxtamembrane domain.[7] Endogenous ligands include epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, amphiregulin, heparin-binding EGF, and betacellulin, although the two most important stimulatory ligands are EGF and TGF-alpha.[8-10]

Binding of ligand leads to dimerization of the receptor with another HER molecule, followed by autophosphorylation of intracellular tyrosine residues. Signal transduction cascades via the Ras, ERK1/2, PI3K/Akt, and STAT pathways are activated. Cellular proliferation, adhesion, differentiation, angiogenesis, and apoptosis result.[6,8,10]

Pharmacologic inhibition of EGFR signaling can be achieved through competitive inhibition of ligand-binding, or by direct tyrosine kinase inhibition. Both of these approaches have undergone extensive clinical development. Two monoclonal antibodies that interfere with ligand-binding have been approved for use in metastatic colorectal cancer-cetuximab (Erbitux) and panitumumab (Vectibix).

Cetuximab

Cetuximab (C225, IMC-225) was developed as a human-murine chimeric antibody derived from the murine antibody M225, which binds specifically to the ligand-binding domain of EGFR.[11] Cetuximab-bound EGFR is not available for binding by natural ligand, and ligand-dependent signaling is reduced. Antibody-bound receptor is internalized, albeit by an alternative and possibly slower process than is ligand-bound receptor.[12] Cetuximab upregulates expression of the cell-cycle inhibitor p27(kip1), resulting in G1 arrest.[13] Reduced proliferation, substantial antitumor effect, and enhanced apoptosis have been demonstrated in cetuximab-treated A431 vulvar carcinoma xenografts.[14] The murine antiepidermal growth factor antibody M225, from which cetuximab was derived by chimerization, substantially enhanced the antitumor effects of doxorubicin in established xenografts of EGFR-expressing tumor cells.[15]

Phase I testing of cetuximab established that therapy with cetuximab is well-tolerated, with acneiform rash and hypersensitivity reactions as the predominant toxicities.[16] Subsequently, hypomagnesemia has also been recognized as an adverse effect of cetuximab.[17] The recommended phase II doses were a loading dose of cetuximab at 400 mg/m² in week 1, followed by a maintenance dose of 250 mg/m² given weekly. Clearance was saturated at this dose and schedule, but an optimum biologic dose was not demonstrated, formal determination of the maximum tolerated dose was not achieved, and alternative schedules such as every-other-week dosing are only now being explored.

Cetuximab in Chemotherapy-Refractory Disease

The phase II experience with cetuximab in colon cancer began with testing in patients with irinotecan-refractory disease, at a time when oxaliplatin was not commercially available in the United States. Patients with disease progression on irinotecan-containing therapy continued the dose and schedule of irinotecan given at the time of progression, with the addition of cetuximab.[18] Objective responses were observed in 22% of patients.

This finding was later confirmed in a large randomized phase II study by Cunningham and colleagues, in a similar population of irinotecan-refractory patients.[19] A total of 329 such patients were randomly assigned to receive either cetuximab and irinotecan (218 patients) or cetuximab monotherapy (111 patients). Objective responses were observed in 22.9 % (confidence interval [CI] = 17.5%-29.1%) of patients treated with the combination, compared with 10.8% of those who received cetuximab alone (CI = 5.7%-18.1%, P = .007). Median time to progression was 4.1 months for patients who received the combination, compared with 1.5 months for those who received the monotherapy (P < .001). Median overall survival was 8.6 months in the combination-therapy group and 6.9 months for patients who received cetuximab alone (P = .48).

Similar objective response rates for cetuximab monotherapy were described in two additional studies: a response rate of 9% among 57 patients with irinotecan-refractory metastatic colorectal cancer treated in a multicenter study, and of 11.6% among 346 patients with irinotecan- and oxaliplatin-refractory disease.[20,21]

A randomized trial of irinotecan with or without cetuximab for patients with metastatic, fluoropyrimidine- and oxaliplatin-refractory colorectal cancer has been completed. The study randomized 1,298 patients between irinotecan and irinotecan/cetuximab. Confirmed responses are reported in 4% of irinotecan, and 16% of irinotecan/cetuximab-treated patients. Median progression-free survival was 2.6 months for irinotecan and 4.0 months for irinotecan/cetuximab (hazard ratio [HR] = 0.69, stratified log-rank P < .0001). However, these improved initial outcome measures were not matched by a comparable advance in overall survival: Median survival was 10.0 months for irinotecan-treated patients and 10.7 months for patients who received irinotecan/cetuximab (P = .7). There was no clear explanation for this discrepancy-one might hypothesize that the survival impact of cetuximab at the time of progression in the control patients was sufficient for survival to match that seen in patients assigned to early cetuximab. The possibility of postprogression acceleration of disease also cannot be excluded on the basis of these data.[22]

Likewise, a comparison of cetuximab with best supportive care for patients with chemotherapy-refractory colon cancer has also been completed, by the National Cancer Institute of Canada (NCIC) and the Australasian Gastrointestinal Trials Group. Patients who had progressed after (or were not candidates for) treatment with a fluoropyrimidine, oxaliplatin, and irinotecan received cetuximab monotherapy or best supportive care, following random assignment. The median survival for patients receiving best supportive care was 4.6 months, compared with 6.1 months for those receiving cetuximab (HR = 0.77, P = .005).[23]

Activity of the cetuximab/irinotecan combination has also been demonstrated when the agents are given on an every-other-week schedule.[24] In a phase II study enrolling patients who had disease progression after treatment with fluorouracil, irinotecan, and oxaliplatin-without requiring evidence of EGFR expression-cetuximab was given as a loading dose of 400 mg/m² in week 1, 250 mg/m² in week 2, and then 500 mg/m² every second week. A response rate of 23% and a median time to progression of 4.7 months were observed in 40 patients. These results are similar to the response and progression data obtained with weekly cetuximab added to irinotecan in the Cunningham study described above.[19]

Cetuximab in Front-Line Combinations

Cetuximab has been added to front-line systemic treatment of metastatic colorectal cancer in a number of studies. European investigators screened 62 patients with unresectable, metastatic colorectal cancer for EGFR expression, and enrolled 43 of the 52 patients with EGFR-positive cancers to a trial of FOLFOX4 chemotherapy (5-FU, leucovorin, oxaliplatin) together with cetuximab.[25] Radiographs were reviewed by an independent response evaluation committee, and the response rate was 77%, including four patients (9%) who had a complete response. The median progression-free survival was 12.3 months, and median survival, 30 months (CI = 17.8-33.8), with a median follow up of 30.5 months. Nine patients were able to undergo R0 resection of metastatic disease after receiving FOLFOX4 plus cetuximab. Grade 3/4 toxicity included diarrhea in 26% of patients, neutropenia in 23%, paraesthesiae in 19%, and grade 3 rash in 28%.

The final results of a randomized phase II study conducted by the Arbeitsgruppe Internistische Onkologie Colorectal Cancer Study Group investigators from Germany were presented in early 2007.[26] A total of 92 patients with measurable metastatic colorectal cancer received capecitabine (Xeloda) and cetuximab, with a random assignment to irinotecan or oxaliplatin. Approximately 70% of the patients had EGFR-expressing tumors. Objective responses, by Response Evaluation Criteria in Solid Tumors (RECIST), were 42% for the irinotecan arm and 66% for the oxaliplatin arm. Progression and survival data are not yet mature for this study.

Cancer and Leukemia Group B (CALGB) 80203 was initially designed as a 2,200-patient study, randomizing patients between FOLFIRI (leucovorin, 5-FU, irinotecan) and FOLFOX, and between the use of cetuximab or not, in a 2?2 design. However, upon the acceptance of bevacizumab as a part of standard first-line therapy, the study closed, having accrued 238 patients.[27] Cetuximab contributed to a modest increase in grade 3 diarrhea and neutropenia among FOLFIRI-treated patients. Objective responses were observed in 36% of patients treated with FOLFIRI, similar to the 40% response rate observed in patients who received front-line FOLFOX without cetuximab. Pooling the chemotherapy arms of the CALGB study, the overall response rate to chemotherapy without cetuximab was 38%, and with cetuximab, it was 52% (P = .029). The addition of cetuximab added to the response rate in each case. The FOLFIRI-plus-cetuximab response rate was 44%, and the FOLFOX-plus-cetuximab response rate was 60%. These results are quite similar to those for cetuximab plus irinotecan or oxaliplatin in combination with capecitabine in the German study cited above.

Predictors of Response and Resistance

Predictors of response and resistance to cetuximab have been eagerly sought, because cetuximab is an expensive drug, usually given on an inconvenient weekly schedule, with unpleasant side effects such as rash and hypomagnesemia, as well as the potential for life-threatening hypersensitivity reactions. Predictive markers would allow us to spare patients who will not benefit from the drug from these expenses, risks, and inconveniences. Perhaps such markers would also point the way to targets for novel therapies that could sensitize tumors to cetuximab.

• EGFR-The first marker to be considered was EGFR. Early studies required immunostaining for EGFR on a tumor sample for enrollment, and this requirement was also included in the US Food and Drug Administration (FDA) indication for cetuximab. The randomized trial of cetuximab or cetuximab plus irinotecan in irinotecan-refractory cancer required immunohistochemical evidence of EGFR expression. A relationship between EGFR expression and outcome was explored in this study: Response rates did not significantly differ for the cetuximab-plus-irinotecan-treated patients by EGFR expression density (categorized as < 10%, 10%-20%, 20%-35%, and > 35% of cells staining, with 50% of cases demonstrating < 10% staining), or by staining intensity (expressed as "faint," "weak or moderate," or "strong").[19]

Pharmacy records were used to identify 16 irinotecan-refractory patients who received cetuximab for colorectal cancer at Memorial Sloan-Kettering Cancer Center who had an EGFR-negative tumor by immunostain. Four objective responses were seen (response rate = 25%).[28] Thus, in colorectal cancer, testing of single archival samples, analysis of the primary tumor in patients with metastatic disease, and the use of immunohistochemical assays that fail to detect low-level EGFR expression all limit our ability to rule out a role for cetuximab in patients with negative EGFR stains.

Since pathologist interpretation of an immunostain for EGFR has not been predictive, it has not been clear whether EGFR measurement is simply unhelpful, whether improved EGFR quantitation is required, or whether EGFR mRNA content, DNA sequence, or gene copy number would be more highly predictive. A small study reported in 2005 suggested that gene copy number would prove to be useful.[29] Eight of nine patients with objective responses who were assessable by fluorescence in situ hybridization (FISH) had an increased EGFR copy number, whereas only 1 of 21 nonresponders assessable by FISH had an increased EGFR copy number (P < .0001). This finding has not yet been clearly reproduced.

With the use of chromogenic in situ hybridization (CISH) to study over 100 colorectal cancers, gene amplification of more than five copies per nucleus was identified in only 12% of primary and 8% of metastatic tumors.[30] Over 80% of these cases demonstrated EGFR immunostaining, and the researchers found poor correlation between immunohistochemical EGFR expression and gene copy number. Studies are ongoing to explore the relationships between cetuximab sensitivity and ligand content, EGFR intron 1 polymorphisms, expression of HER2, HER3, or HER4, and downstream signals such as ras or pAkt.

• Dermatologic Toxicity-To date, the strongest predictor of cetuximab sensitivity has been the development of the characteristic dermatologic toxicity.[19] The most striking manifestation of this is an erythematous, acneiform rash, distributed predominantly over the face, scalp, and trunk. This may be extensive, with pain and burning, or there may be only scattered follicles involved. The individual lesions may be large and cystic, or very small and circumscribed around the follicle. Dry skin, scaling, ichthyosis-like changes, paronychia, fissuring of the skin of the fingers and toes, and trichomegaly are also seen.[31] Biopsy reveals an early round cell infiltrate, with the development by day 4 or 5 of neutrophilic folliculitis, which is usually sterile. Anecdotal evidence suggests that anti-inflammatory strategies may have efficacy in managing the skin toxicity,[32] and such approaches are currently being studied.

The question has arisen as to whether rash is a surrogate for adequate dosing. Given the relationship between rash and survival, and the possibility that rash appears only when significant binding of EGFR in skin occurs, would patients who do not initially develop a rash benefit from a higher dose of cetuximab?

A pilot study has addressed this question in patients with irinotecan-treated and progressive metastatic colorectal cancer.[33] Patients were treated with a cetuximab/irinotecan combination, and rash was assessed on day 22. Patients who did not have moderate to severe rash by day 22, and whose disease was nonprogressive, were randomly assigned to continue on the standard maintenance dose of cetuximab, or to receive escalations in cetuximab dose in 50 mg/m² increments, until a maintenance dose of 500 mg/m² had been reached. The overall incidence of skin toxicity was increased to 50% in patients who received the higher doses of cetuximab, compared with 36% in patients who continued on the standard dose. The partial response rate was also slightly increased, reaching 30% for patients receiving escalated-dose cetuximab, compared with 13% for the standard-dose arm. This was offset by a slightly lower number of patients with stable disease, so that the number of patients who had disease progression or were unevaluable was similar in the two groups.

Panitumumab

The clinical success of EGFR targeting with cetuximab has led to interest in alternative agents that also interfere with ligand activation of EGFR signaling, perhaps with greater activity or more favorable toxicity profiles or at lesser cost. Panitumumab (clone E7.6.3, ABX-EGF) is a fully human, IgG2 antibody, which is also approved for the treatment of chemotherapy-refractory colorectal cancer. This antibody is raised in mice that are genetically engineered without functional murine immunoglobulin expression and into which human immunoglobulin genes have been introduced.

Panitumumab binds EGFR with an affinity of 5 ? 10-11, blocks binding of EGF and TGF-alpha to the receptor, and inhibits EGFR phosphorylation.[34] The affinity of panitumumab for EGFR is thus greater than that of cetuximab; the biologic consequences of panitumumab-binding appear similar to those of cetuximab-binding. Like cetuximab, panitumumab has antitumor activity in a variety of cell lines, and its effect is additive with chemotherapy in A431 squamous cell carcinoma cells.

The phase I study of panitumumab was conducted in patients with 2+ to 3+ staining intensity for EGFR on at least 10% of tumor cells.[34] Forty-three patients with solid tumors were treated. The agent caused an acneiform rash in a dose-dependent manner. One patient with prostate cancer had a serologic response, and one patient with colorectal cancer had a partial response. No human anti-human antibody formation or hypersensitivity reaction was demonstrated.

A large dose-escalation trial in patients with renal cell cancer demonstrated a 100% incidence of rash, characterized as transient, at a dose of 2.5 mg/kg/wk. The authors commented that this incidence of rash is indicative of full-receptor occupancy-a reasonable hypothesis, although it is important to note that the specific correlation of occupancy of skin receptors to occupancy of tumor receptors may differ among antibodies of different affinities. Skin biopsy-to-tumor biopsy comparisons or nuclear imaging of the distribution of radiolabelled antibody would be required to answer this question more definitively.

Phase II Experience in Refractory Colon Cancer

Panitumumab has been evaluated in separate studies for patients with EGFR-negative/poor and EGFR-rich, chemotherapy-refractory colon cancer. The study in EGFR-negative or -poor cancers enrolled patients in whose tumors no, or 1% to 9%, of cells stained for EGFR by immunohistochemistry.[35] Panitumumab was given at a dose of 6 mg/kg every second week. An interim analysis was reported after 88 patients had enrolled, and 23 patients had 20 weeks of follow-up. Low-grade skin toxicity and diarrhea were the most common toxicities. Hypomagnesemia was seen in 8%. Three patients had hypersensitivity reactions, of which one was high-grade and led to treatment discontinuation. Only the patients with 20 weeks of follow-up were considered in the efficacy analysis. Objective responses were seen in 18% of EGFR-negative and 8% (1 patient) with EGFR-poor cancers, respectively.

Interim results were presented from a parallel study, which had enrolled 91 patients with EGFR immunostaining of ≥ 10% of cells.[36] Efficacy data were available on 39 patients, of whom 8% had a partial response. Toxicity data were similar, with one grade 3 hypersensitivity reaction.

Phase III Experience in Refractory Colon Cancer

The FDA approval of panitumumab followed presentation of the data from a randomized phase III trial of panitumumab vs best supportive care in patients with chemorefractory metastatic colorectal cancer.[37] The study randomized 463 patients to either best supportive care or to panitumumab at 6 mg/kg every other week. Because of the high frequency of panitumumab-induced rash, it was not possible to blind the treatment assignment in this study, and crossover to panitumumab was permitted for patients progressing on best supportive care who had received no other anticancer therapies since enrollment. About 76% of patients initially assigned to best supportive care crossed over to panitumumab upon progression.

The progression-free survival curves diverge early, with many patients progressing prior to the first planned tumor reassessment. At 8 weeks, 49% of panitumumab-treated and 30% of best supportive care patients were progression-free. Because of the possible incentive for early restaging provided by the potential for crossover, the more informative portions of the progression curves are those following 8 weeks, where an advantage for panitumumab is also seen. Objective responses were seen in 8% of patients receiving panitumumab as the initial treatment assignment, 0% of those receiving best supportive care, and 12% of those receiving panitumumab in the crossover portion of the study. The median time to progression after crossover was 8 weeks.

There was no survival advantage to initial treatment with panitumumab. Given the high proportion of patients who crossed over, and the likelihood that they represent a biologically different subset than those who were unable to crossover, it will not be possible to determine definitively from this study whether there is a survival advantage to the use of panitumumab in the chemotherapy-refractory setting, although it seems likely that crossover was the reason a survival advantage did not emerge.

Phase II Experience in Front-Line Therapy

Panitumumab has also been tested in combination with bolus IFL (5-FU, leucovorin, irinotecan; Saltz regimen) or FOLFIRI for first-line treatment of metastatic colorectal cancer.[38] Patients were only eligible if there was EGFR expression in > 10% tumor cells and no prior chemotherapy had been given for metastatic disease. A total of 43 patients were enrolled. Grade 3/4 diarrhea was seen in 39% of patients and seemed higher in IFL recipients than in FOLFIRI-treated patients. Skin toxicity was seen in all patients but was grade 3 in only 17%. Partial response was seen in 39% of patients. Progression and survival data were not mature at the time of the report.

Things We Do Not Know

No evidence suggests that either drug would have activity after a cancer has progressed on the other. Thus, substitution of panitumumab for cetuximab after a hypersensitivity reaction is a reasonable course, but trial of one drug after progression on the other cannot currently be justified.

In the laboratory, the most striking difference between the drugs relates to the higher affinity of panitumumab for the receptor. This was expected to lead to better efficacy, but the data available at present do not suggest a therapeutic advantage for one agent over the other, and efficacy data for cetuximab are more extensive. Recent laboratory work has suggested that single-chain antibodies of high affinity have more restricted tumor penetration than moderate affinity antibodies, perhaps because they are so readily bound by the first targets they encounter on leaving the circulation.[39] The implications of this finding for the clinical development of antibodies to targets within the tumor (as opposed to within the tumor vasculature) remain unclear.

Comparison of Cetuximab and Panitumumab

Cetuximab and panitumumab are highly comparable drugs with similar indications. No studies have compared the agents head-to-head in patients. Thus, any comparison between the agents must come from results of different studies. There is far more information available on cetuximab, which has undergone testing for longer and been on the market longer. Even the efficacy of panitumumab is relatively poorly understood relative to that of cetuximab.

It seems the toxicity profile favors panitumumab, given a lower rate of high-grade hypersensitivity reactions with panitumumab. Cost also currently favors panitumumab on a per month basis, but no cost-benefit analyses based on different assumptions about relative efficacy and duration of therapy have been conducted. The dosing schedule currently favors panitumumab, but ongoing studies are exploring every-other-week schedules with cetuximab as well.

At present, therapeutic use will likely be guided by the clinical situation. Data on the use of cetuximab with chemotherapy are robust, so that for patients with irinotecan-refractory cancer who continue to be candidates for cytotoxic chemotherapy, the combination of irinotecan and cetuximab-with its higher response rate and longer time to progression than reported for either panitumumab or cetuximab monotherapy-would be the appropriate choice. For patients with similar chemotherapy refractoriness who are not fit for further chemotherapy, and for whom an antibody alone is appropriate, panitumumab and cetuximab currently appear to be therapeutically similar choices, with some cost, safety, and convenience advantages to panitumumab, but with the only clear survival data coming from the NCIC study of cetuximab.

The use of a monoclonal antibody to EGFR is sometimes considered in first-line treatment (in combination with chemotherapy) for patients who are poor candidates for bevacizumab because of recent arterial thrombotic events. For the present, more data support the activity of cetuximab than of panitumumab in this setting. It would be reasonable to choose cetuximab for such a patient and reserve panitumumab in the event of hypersensitivity to cetuximab.

Ongoing studies, including randomized trials in first-line treatment, trials of these antibodies in combination with antiangiogenic therapies, and extensive correlative science efforts directed toward improving patient selection, will undoubtedly refine our use of both agents in the near future.

Disclosures:

Dr. Burtness is an advisor for Sanofi-Aventis, Amgen, Genentech, ImClone Systems, and Array BioPharma; a speaker for Sanofi, Genentech, and ImClone; and has received research funding from Sanofi, Bristol-Myers Squibb, and Genentech.

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