This meta-analysis was performed to determine whether the addition of monoclonal antibodies (mAbs) of epidermal growth factor receptor (EGFR) to oxaliplatin-based chemotherapy treatment improves efficacy in KRAS wild-type metastatic colorectal cancer (mCRC), and whether infusional 5-fluorouracil (5-FU) and oxaliplatin is a preferred combination for EGFR mAbs. Oxaliplatin (including treatment), EGFR mAbs, first-line treatment, KRAS wild-type, and mCRC were used as key words. The PRIME, OPUS, COIN, and NORDIC VII trials were identified by two independent authors. Time-to-event outcomes of overall survival (OS) and progression-free survival (PFS) were analyzed using HRs (hazard ratios) with fixed effect, and response rate (RR) using odd ratios (OR) with fixed effect. A total of 1767 patients who were KRAS wild-type were included in this meta-analysis, with 866 patients in the mAbs and chemotherapy combination group and 901 patients in the chemotherapy alone group. The addition of mAbs to oxaliplatin-based chemotherapy in patients with KRAS wild-type mCRC as first-line treatment resulted in significant improvements in PFS (HR = 0.88; 95% confidence interval (CI), 0.79–0.99; P = 0.03) and response rate (RR) (OR = 1.38; 95% CI, 1.14–1.66; P =0.009) compared with chemotherapy alone, but the difference in OS was not significant (HR = 0.96; 95% CI, 0.85–1.08; P = 0.48). However, the differences in OS and PFS were not significant when mAbs were added to bolus 5-FU or capecitabine-based regimens compared with chemotherapy alone, whereas PFS improved with an infusional 5-FU and oxaliplatin combination (P = 0.06; PFS, HR = 0.76; 95% CI, 0.65–0.86; P = 0.0002), and even OS was marginally significant, which was consistent with the subgroup analysis of cetuximab and panitumumab. EGFR mAbs combined with oxaliplatin and an infusional 5-FU regimen was associated with significantly improved RR, PFS and OS as first-line treatment in KRAS wild-type mCRC.
Colorectal cancer (CRC) ranks as the third most common cancer in males and as second in females worldwide and is the fourth leading cause of cancer death.[1, 2] The prognosis of metastatic colorectal cancer (mCRC) has improved over the last several decades, which is largely attributed to a result of better use of fluoropyrimidine regimens, including the addition of folinate, and the combination with chemotherapeutic agents such as irinotecan and oxaliplatin, as well as the development of some new targeted drugs.
It is noteworthy that overexpression of epidermal growth factor receptor (EGFR) and its ligands, such as EGF and transforming growth factor α (TGFα), has been identified in nearly 25% to 77% of CRC cases, which is associated with more aggressive disease, poor prognosis in mCRC, and even chemoresistance.[4-7] Therefore, targeting the EGFR pathway with monoclonal antibodies (mAbs) seems to be a promising therapeutic strategy.
However, evidence from clinical practice suggests that EGFR overexpression is not a useful predictive biomarker, while KRAS genotype plays a key role in this signaling pathway. Across studies, treatment with anti-EGFR mAbs produced better survival in patients with KRAS wild-type, whereas these drugs had no effect in patients with mutant KRAS. Furthermore, the response rate (RR) of EGFR mAbs in patients with KRAS wild-type was increased even in trials that did not reach their time-related end points of overall survival (OS) or progression-free survival (PFS).
Since EGFR mAbs demonstrate significant benefit in KRAS wild-type mCRC as a single regimen or in combination with standard chemotherapy, such as oxaliplatin- or irinotecan-based treatments, the US Food and Drug Administration approved the clinical use of cetuximab (Cmab) and panitumumab (Pmab) in 2004 and in 2006, respectively.[9-15]
Some earlier studies have shown that high EGFR expression has a relationship with chemotherapy resistance to various traditional cytotoxic agents, including oxaliplatin, and the results still confirm that gene polymorphisms of EGFR and its downstream effector, interleukin-8 (IL-8), predict oxaliplatin efficacy in patients with advanced CRC.[16, 17] Preclinical work proved that, with oxaliplatin treatment, intracellular reactive oxygen species (ROS) are generated, and ROS potently activates Src. In oxaliplatin-resistant cell lines, Src activity is constitutively increased, and they can disregard the EGFR-dependent cell growth and survival signaling pathway, resulting in an undesired effect of EFGR mAbs combined with oxaliplatin-based chemotherapy.[18, 19] Hence, oxaliplatin is frequently questioned as a partner for EGFR in the treatment of mCRC.
Recently, Skvortsov et al. found that Cmab is able to suppress the expression of thymidylate synthase (TS), which is involved in the mechanism of 5-FU action. Combined treatment with Cmab and 5-FU revealed a synergistic anti-tumor response that is closely correlated with functional activity of EGFR/MAPK pathway. Later, a research published in Cancer Science showed the molecular mechanism underlying the synergistic interaction between trifluorothymidine (TFT) and the EGFR inhibitor erlotinib in human colorectal cancer cell lines. The results suggested that Erlotinib inhibited TS activity in EGFR-expressing cell lines, probably due to cell cycle arrest in the G1 phase. And TS activity was lower in the combinations; probably a result of cell cycle interference.
Several multi-center, randomized, controlled, clinical trials have shown puzzling findings about whether efficacy is improved by the addition of mAbs of EGFR to oxaliplatin-based chemotherapy in KRAS wild-type mCRC.[13, 21-24] Hence, the National Comprehensive Cancer Network guidelines suggest that Cmab should no longer be combined with oxaliplatin, whereas Pmab could be added to FOLFOX according to the results of the PRIME trial.[9, 13] This meta-analysis aimed to determine whether efficacy is improved by the addition of EGFR mAbs to oxaliplatin-based chemotherapy as first-line treatment in patients with KRAS wild-type mCRC, and whether infusional 5-fluorouracil (5-FU) is a preferred backbone for EGFR mAbs in this combination.
Materials and Methods
Literature search strategy
Oxaliplatin (including treatment), EGFR mAbs, first-line treatment, KRAS wild-type, mCRC, and random were used as medical subject headings for the searches. Databases searched up to September 2012 were: Ovid Medline (1946–2012), PubMed (1966–2012), Science Citation Index Expanded (1950–2012), Embase (1988–2012), American Society of Clinical Oncology (ASCO; 1996–2012), ASCO Gastrointestinal Cancers Symposium (1996–2012), Cochrane Library (1950–2012), Cochrane Methodology Register (2004–2012), Cochrane Central Register of Controlled Trials (2004–2012), Cochrane Database of Systematic Reviews (2005–2012), Database of Abstracts of Reviews of Effects (2004–2012), Health Technology Assessment (2004–2012), and National Health Service Economic Evaluation Database (2004–2012).
Inclusion and exclusion criteria
Clinical studies identified from the databases according to the literature search strategy that included basic requirements for a meta-analysis based on hazard ratios (HRs) were eligible for the analysis. Time-to-event outcomes of OS and PFS were analyzed as the primary events, and the secondary event was RR. Redundant studies that were irrelevant, gave no information about patients' KRAS status, included other mAbs, such as bevacizumab, not oxaliplatin-based chemotherapy, or included second- and third-line treatment for mCRC were excluded. Reports that were editorials, comments, reviews, or with no results for KRAS wild-type patients were also excluded.
All relevant characteristics were collected, including patient demographics, clinical pathology, KRAS status, oxaliplatin-based chemotherapy treatment, and clinical outcome of EGFR mAbs combination treatments. Targeted data were extracted by two independent authors to ensure the quality of the meta-analysis. PFS was defined as the time between randomization and first documented progression, death, or last follow-up. OS was defined as the time from randomization to death or last follow-up. RR was the percentage of patients with complete or partial responses based on the patients' imaging evaluations (i.e., the Response Evaluation Criteria in Solid Tumors, or RECIST) after at last three cycles of treatment.[25, 26]
Revman 5.1.7 software was downloaded from the Cochrane Collaboration and used for the meta-analysis. Time-to-event outcomes of OS and PFS were analyzed using HRs with fixed effect, and RR using odd ratios (OR) with fixed-effects model (the Mantel-Haenszel method) for a high heterogeneity. The O-E, Variance, HR, lnHR, and variance of the lnHR could be generated according to the HR and its associated confidence interval (CI) from reported summary statistics.[28, 29] A two-sided P < 0.05 was considered significant.
Only the fixed-effect model was used for the analysis.[30, 31] The heterogeneity among studies was explored using the χ2 test with significance set at P < 0.100, and it was quantified using I2, with a maximum value of 30% for low heterogeneity.[30-33] Exp [(O-E)/var] was used as the statistical method.[28, 33, 34]
A subgroup analysis of the addition of mAbs to infusional 5-FU, bolus 5-FU, or capecitabine-based regimens was performed for the outcomes of OS, PFS and RR to identify the differences among the three kinds of fluoropyrimidine backbones and to verify the preferred chemotherapy partner with oxaliplatin for mAbs of EGFR.
Publication bias was identified with funnel plots,[29, 35] whereby asymmetries in the funnel plot indicated publication bias. The risk of included studies bias was assessed in accordance with the guidelines of the Cochrane Collaboration.[36-38]
Characteristics of the trials
Four trials with usable information met the inclusion criteria found through searching the databases and related references. Trials in this meta-analysis were PRIME (Pmab randomized trial in combination with chemotherapy for mCRC), OPUS (oxaliplatin and Cmab in first-line treatment of mCRC), COIN (continuous chemotherapy plus Cmab, or intermittent chemotherapy with standard continuous palliative combination chemotherapy treatment with oxaliplatin and a fluoropyrimidine in the first-line treatment of mCRC), and NORDIC VII (FLOX regimen given continuously or intermittently, in combination with Cmab in first-line treatment of mCRC). All of the included trials were of high quality, and the efficacy of mAbs was re-analyzed with the interaction of KRAS status. A total of 3716 patients were analyzed in the four trials, but the total number of patients included in this meta-analysis was 1767; all were KRAS wild-type, with 866 patients in the mAbs and chemotherapy combination group and 901 patients in the chemotherapy alone group. The characteristics of these studies are shown in Table 1.
All patients in the four trials were mCRC at the time of first-line chemotherapy treatment, which was FOLFOX4 in the PRIME and OPUS trials, XELOX/FOLFOX in the COIN trial, and FLOX in the NORDIC VII trial.[13, 21-23] The patients' characteristics are shown in Table 2. A total of 1767 patients was given oxaliplatin-based chemotherapy. Patients in the mAbs and chemotherapy combination group were given EGFR mAbs Cmab (OPUS, COIN, and NORDIC VII) and Pmab (PRIME). In the three trials, Cmab was given as an initial intravenous dose of 400 mg/m2, followed by 250 mg/m2 once a week. Pmab was administered as 6 mg/kg every 2 weeks on day 1 before FOLFOX4 treatment in the PRIME trial.
ECOG, Eastern Cooperative Oncology Group; NA, not available; PS, performance status; WHO, World Health Organization.
Site of primary tumor
Number of metastatic sites
PFS and OS of mAbs and chemotherapy combination versus chemotherapy alone
Four trials presented Kaplan–Meier curves and HR and CI results for PFS and OS in 1767 KRAS wild-type cases, of which 1111 patients were treated with the Cmab combination, and 656 patients were treated with Pmab.
The addition of mAbs to oxaliplatin-based chemotherapy in patients with KRAS wild-type mCRC as first-line treatment resulted in significant improvements in PFS (HR = 0.88; 95% CI, 0.79–0.99; P = 0.03; Fig. 1) compared with chemotherapy alone, but the difference in OS was not significant (HR = 0.96; 95% CI, 0.85–1.08; P = 0.48; Fig. 2), indicating that the addition of mAbs to oxaliplatin-including chemotherapy lengthened the interval to disease progression, but showed no significant improvement in long-term efficacy.
To further understand the specific agent of mAbs, a subgroup analysis of Cmab and Pmab was performed. The PFS of Cmab combined with chemotherapy showed no significant difference (HR = 0.93; 95% CI, 0.81–1.06; P = 0.26; Fig. 1), whereas the Pmab combination group had an improved PFS (HR = 0.80; 95% CI, 0.66–0.97; P = 0.02; Fig. 1), and the difference between the Cmab and Pmab groups was not significant (P = 0.22). The OS in both groups were not significantly different between the Cmab or Pmab and chemotherapy combination compared with chemotherapy alone (HR = 1.02; 95% CI, 0.89–1.18; P = 0.50 for Cmab; HR = 0.83; 95% CI, 0.67–1.02; P = 0.08 for Pmab; Fig. 2), and the difference between the Cmab and Pmab groups was not significant (P = 0.11). Both the PFS and OS results were not completely consistent with the subgroup analysis.
PFS and OS of mAbs combined with chemotherapy versus chemotherapy alone in patients treated with different fluoropyrimidines
To determine the reason why both PFS and OS results were not completely consistent with the subgroup analysis, and whether the mode of fluoropyrimidine administration played an important role in the efficacy improvement of mAbs and oxaliplatin-based chemotherapy, subgroups of different fluoropyrimidine administration modes were analyzed.
Surprisingly, the differences in OS and PFS were not significant when mAbs were added to bolus 5-FU (OS, HR = 1.14; 95% CI, 0.80–1.62; P = 0.46; PFS, HR = 1.07; 95% CI, 0.78–1.46; P = 0.67) or capecitabine-based regimens (PFS, HR = 1.06; 95% CI, 0.88–1.28; P = 0.54) compared with chemotherapy alone, whereas PFS improved with an infusional 5-FU and oxaliplatin combination (PFS, HR = 0.76; 95% CI, 0.65–0.86; P = 0.0002; Fig. 3), and OS was marginally significant (OS, HR = 0.86; 95% CI, 0.74–1.00; Fig. 4). The difference among the three subgroups was significant (P = 0.010) when the PFS of mAbs combined with chemotherapy versus chemotherapy alone in patients treated with different fluoropyrimidines was analyzed.
Furthermore, in order to eliminate different therapeutic effects of Cmab and Pmab in infusional 5-FU and oxaliplatin combination, a subgroup was performed. PFS of both Cmab and Pmab groups were improved (Cmab, HR = 0.70; 95% CI, 0.56–0.88; P = 0.002; Pmab, HR = 0.80; 95% CI, 0.66–0.97; P = 0.02; Fig. 5), while differences of OS were not significant (Cmab, HR = 0.90; 95% CI, 0.72–1.13; P = 0.36; Pmab, HR = 0.83; 95% CI, 0.67–1.02; P = 0.08; Fig. 6). Neither subgroup difference of PFS nor OS was significant (PFS: P = 0.39; OS: P = 0.62). The differences of infusional 5-FU and oxaliplatin combination were still statistically significant without the influence of Cmab and Pmab.
The results suggested that the infusional 5-FU increased the PFS of the mAbs and oxaliplatin-based chemotherapy combination group, along with a trend to increase OS.
RR of mAbs and chemotherapy combination versus chemotherapy alone
The difference in RR of mAbs and chemotherapy combination versus chemotherapy alone was significant (OR = 1.38; 95% CI, 1.14–1.66; P =0.009, Fig. 7), especially combined with Cmab (OR = 1.41; 95% CI, 1.11–1.79; P =0.005). Test for subgroup difference of Cmab and Pmab showed I2 = 0%, P = 0.76, verifying the effect of mAbs in combination of oxaliplatin including chemotherapy in the first-line treatment of KRAS wild-type mCRC.
Bias of inclusion studies
To evaluate the bias risk in the studies, sequence generation, allocation concealment, blinding of participants (personnel and outcome assessors), incomplete outcome data, selective outcome reporting, baseline imbalance, early stopping, academic, and source of funding were collected from the original studies.[25, 30, 31] The four trials were of high quality because they were multi-center, randomized, controlled, clinical studies.
A funnel plot was performed to analyze publication bias, and it did not show any publication bias when the four trials were added into the analysis.[39, 40] Unfortunately, there were only four studies, so the criteria to explore publication bias with Egger's test were not met.
Until recently, Cmab and Pmab, two classical EGFR mAbs, have shown benefit in the treatment of advanced CRC both as single agents and combined with conventional chemotherapy, as confirmed by several randomized trials, and they have been approved by North American and European regulatory agencies.[41-44] Cetuximab (Cmab) is a recombinant human/mouse chimeric immunoglobulin (Ig) G1 that specifically binds the extracellular domain II of EGFR. It competes with EGFR ligands, such as EGF or TGFα, with a high affinity (Kd = 1 × 10−10 M).[6, 7] The major mechanisms for the cytostatic action of Cmab are that it directly inhibits EGFR signaling and activates the immune system called antibody-dependent cellular cytotoxicity (ADCC). Panitumumab (Pmab) is a fully human immunoglobulin G2 (IgG2) mAb with a high affinity for EGFR (Kd = 5 × 10−11 M).[6, 45] It has been proven that Pmab arrests cell cycle progression and blocks cancer growth, but it does not induce anticancer activity through ADCC because it is an IgG2, in contrast with the IgG1 Cmab. However, the main mechanism of both Cmab and Pmab goes the same way. The binding of these mAbs on EGFR prevents the dimerization and the activation of EGFR. Thus, the ligand-dependent activation of RAS-RAF-MAPK and PI3K-AKT-mTOR pathways is blocked.
Whether there is a preferred partner for EGFR mAbs in the first-line treatment of mCRC is a puzzling question leading to widespread concern, especially with respect to whether oxaliplatin-based chemotherapy is an optimal choice for such a combination. Oxaliplatin, as a third-generation platinum analog that induces DNA cross-links and results in apoptosis, was initially approved for use in the USA in 2001 and is currently approved in both first- and second-line, as well as adjuvant, settings.[46, 47] The conventional oxaliplatin-including chemotherapy was XELOX administered in the COIN trial, FOLFOX in PRIME, OPUS, and COIN trials, and FOLX in the NORDIC VII trial.
From the present meta-analysis, it is important to note that the combination of mAbs and oxaliplatin-based chemotherapy as the first-line treatment of mCRC has shown significant improvements in PFS (HR = 0.88; 95% CI, 0.79–0.99; P = 0.03), without significant improvements in OS (HR = 0.96; 95% CI, 0.85–1.08; P = 0.48). However, both PFS and OS results were not completely consistent with the subgroup analysis. These confused results added another layer to the already complex and to some degree inconsistent outcomes of the clinical trials.
It must be noted that oxaliplatin has very little activity as a single agent in the treatment of CRC, and it depends on fluoropyrimidine to demonstrate efficacy. In this regard, an additional subgroup analysis was performed according to the mode of fluoropyrimidine administration (infusional 5-FU, bolus 5-FU, and capecitabine) in oxaliplatin-based chemotherapy. It became evident that the differences in OS and PFS were not significant when mAbs were added to bolus 5-FU (OS, HR = 1.14; 95% CI, 0.80–1.62; P = 0.46; PFS, HR = 1.07; 95% CI, 0.78–1.46; P = 0.67) or capecitabine-based regimens (PFS, HR = 1.06; 95% CI, 0.88–1.28; P = 0.54) compared with chemotherapy alone, whereas OS and PFS were improved with an infusional 5-FU and oxaliplatin combination (OS, HR = 0.86; 95% CI, 0.74–1.00; P = 0.06; PFS, H =0.76; 95% CI, 0.65–0.86; P = 0.0002).
As we know, 5-FU has become a mainstay anticancer regimen since it was introduced in 1957, and it has been the only therapy available for mCRC modulated by leucovorin in 1996, which can be administered either as a protracted infusion or as a bolus injection. Then, capecitabine, the first oral fluoropyrimidine approved for mCRC, became available, leading to an armamentarium of treatment for mCRC. We have come to doubt whether the effect of oxaliplatin on EGFR mAbs combination outcome has some relationship with the effect of the specific fluoropyrimidine backbone.
One potential molecular mechanism explanation for this could lie in the fact that the oxaliplatin-based chemotherapy and EGFR mAbs action differs depending on whether 5-FU is administered as a bolus injection or as a protracted infusion. The most important mechanism for 5-FU bolus schedules is incorporation of fluorouridine triphosphate into RNA. As the infusion time is longer, the inhibition of thymidylate synthase (TS) becomes significantly more important.[9, 49] In addition, some recent studies suggested that EGFR inhibitors can reduce TS expression levels, which have an inverse relationship to the efficacy of 5-FU in CRC.[8, 50] This may demonstrate that infusional 5-FU is a preferred fluoropyrimidine backbone for the oxaliplatin and EGFR mAbs combination, which is consistent with another meta-analysis published recently. It is still unclear why capecitabine failed to improve the EGFR mAbs and oxaliplatin combination. More clinical trials including capecitabine are needed to confirm this result.
There are several limitations in the present meta-analysis. First, because EGFR mAbs have not long been used in clinical practice and only four studies were included in the analysis, limited data weakens the certainty of our findings. Furthermore, the sample sizes of the infusion 5-FU, bolus 5-FU, and capecitabine groups were not large enough to perform sensitivity analyses or meta-regression analysis, which was the most important disadvantage of our study. However, according to the recent NCCN guidelines, the combination of EGFR mAbs and oxaliplatin has been canceled because of the controversial results of clinical trials. There lies little chance of new multi-center clinical trial about this combination. So the four studies analyzed in our study show some kind of special implications in clinical experience. Second, although every effort was made to find all of the relevant information, the OS result of the XELOX and Cmab group in the COIN trial was not found, resulting in a lack of statistical power to analyze the effect of different fluoropyrimidine backbones on the oxaliplatin and Cmab combination, especially the analysis of capecitabine, and it also resulted in a marginal P-value in the subgroup analysis of OS.
Additionally, some studies suggest that mutations in K-ras codons 61 and 146, as well as in the B-raf, N-ras, and PI3K3CA oncogenes, relate to resistance to mAbs in K-ras WT patients, so more high-quality, randomized, clinical trials focusing particularly on other gene mutations are needed to confirm these results.[52, 53]
To the best of our knowledge, though this was not the first meta-analysis that examined whether patients with KRAS wild-type mCRC could benefit from additional EGFR mAbs in the first line oxaliplatin-based chemotherapy setting, we were specifically concerned with whether the additional use of EGFR mAbs to oxaliplatin-based chemotherapy treatment had a better efficacy, which further explained the results reported by two meta-analyses published in 2012.[51, 54] A recent report published in the Journal of Clinical Oncology in 2013 by Le-Chi Ye et al. showed that adding cetuximab to mFOLFOX6 in patients with initially unresectable KRAS wild-type colorectal liver metastases underwent better survival in PFS and OS compared with mFOLFOX6 alone, which was consistent with our research.
With the advantages and disadvantages listed above, the present results indicate that EGFR mAbs combined with oxaliplatin and an infusional 5-FU regimen was associated with significantly improved PFS and OS as first-line treatment in KRAS wild-type mCRC. To better understand the molecular mechanisms involved in the interaction between EGFR mAbs and cytotoxic agents, more preclinical and translational studies are needed.
This work was supported in part by the Project of the National Natural Science Foundation of China (NO: 81071862). The authors thank professors of the Department of Health Statistics of West China School of Public Health and professors of the Department of Evidence-Based Medicine and Clinical epidemiology for their fruitful discussions about the statistical methods used in this study.