In the present study, we collected all available studies and carried out a meta analysis to examine the association of variations of EGFR GCN with prognosis of advanced CRC patients. Ten studies involving 776 patients on OS, eight studies involving 893 patients on PFS and three studies on TTP were critically reviewed. We subgrouped the articles into five groups (ethnicity, KRAS status, anti-EGFR mAbs, detection method and line of treatment). Meta-analysis showed increased EGFR GCN was significantly associated with improved OS and PFS but not TTP. The median OS of patients harboring increased GCN showed 1.61-fold increase, the median PFS showed 1.54-fold increase. A meta-analysis of these studies confirms that increased EGFR GCN is indeed associated with a moderate OS and PFS benefit, from anti-EGFR treatment for metastatic CRC patients. Similarly, EGFR gene copy number has also been evaluated as a potential predictor of response of tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer patients, and a meta analysis has demonstrated an association between increased EGFR copy number, and improved survival outcomes 
. Recently, Yang et al.
performed a meta analysis to differentiate the objective response rate (ORR) between patients with increased EGFR GCN and those with no increased EGFR GCN 
. They suggested a general trend towards higher ORR in patients with increased EGFR GCN. However, for important prognostic factors as PFS and OS, as the data was relatively incomplete, they only descriptively reviewed published papers and did not perform quantitative synthesis of the studies. In this study, several excellent HR extraction methods were used to calculate the pooled HR quantitatively. The result showed increased EGFR GCN association with improved survival outcomes among anti-EGFR-treated patients. These results imply the EGFR GCN might be not only an effective predictive but also a valuable prognostic marker.
The anti–EGFR monoclonal antibody is effective in prolonging survival in patients with metastatic colorectal cancer after failure of conventional chemotherapy 
. In our stratified analysis, the increased EGFR GCN was significantly associated with improved OS and PFS in those populations that received second-line or higher but not first-line, which coincided with the strategies in clinical practice of chemotherapy. From a clinical point of view, not only in the US and Europe but also in China, anti-EGFR mAbs were usually used in wild-type KRAS mCRC patients. So, assessing the role of EGFR GCN in patients with wild-type KRAS may be more meaningful. In this study, we found that the prognostic value of EGFR GCN on survival appears to not be related to KRAS status, which suggested EGFR GCN might be an independent prognostic biomarker. The significant association between survival with EGFR GCN, revealed tumor growth is probably mainly driven by the EGFR pathway and this biological characteristic is evoked by an increase in EGFR copy number.
EGFR is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers two main signaling pathways, the RAS-RAF-MAPK axis, which is mainly involved in cell proliferation, and the PI3K-PTEN-AKT pathway, which is mainly involved in cell survival and motility 
. The anti-EGFR mAbs have been proven to be effective in metastatic colorectal cancer. The molecular mechanisms underlying the clinical response to this drug remain unknown. Genetic alterations in EGFR-related signaling pathways may have an effect on response to this targeted therapy, which may be due to the constitutive activation of the downstream genes of the EGFR signaling pathway such as KRAS, BRAF, or PIK3C2A, or to the loss of a tumor suppressor gene such as PTEN. Until now, the most acceptable marker, as a predictive and prognostic factor, was the status of KRAS. However, KRAS was not the only predictor of the cetuximab response. The present study was aimed at assessing the prognostic role of EGFR GCN, in terms of clinical outcome, in patients treated with anti-EGFR mAbs. EGFR GCN detection also appears to be relevant to positively identify responders. Variations of GCN, reflect the many different routes taken by individual tumors to disrupt/escape mechanisms governing normal cellular behavior. In most solid tumors, including CRC, the best characterized mechanisms underlying increased EGFR GCN are gene amplification and chromosome 7 polysomy 
Current obstacles for a future clinical application of EGFR GCN are mainly concentrated on the following two aspects: detection methods and difficult technical reproducibility. FISH technique has been used in most previous studies, but the FISH results are challenging to interpret and the lack of standardization of analytical methods and scoring systems may partly explain why the EGFR GCN evaluation has not been incorporated into clinical practice yet 
. When looking at the different cutoff values in the literature, we found reproducibility remains a large obstacle for its practical usefulness and an international consensus on the definition of cutoff points is needed. Sartore-Bianchi et al.
also found that molecule diagnosis of EGFR GCN by FISH among five highly experienced pathology centers varied largely, a detailed scoring system and comprehensive training programmes are necessary 
. Although different cutoff points have been applied, 95% CIs around sensitivity and specificity yielded by each cutoff point were similar, thus indicating that results from these studies are consistent. In the present study, we analyzed the influence of GCN detection method on survival and did not found any discrepancies.
There are several limitations kept in consideration in this meta-analysis. First, most of the studies were not conclusive because they evaluated limited patient series that were nonhomogeneously treated. Second, relatively small sample sizes included in East Asians may also inﬂuence the results, and further studies are necessary to detect the potential role of GCN. Third, primarily the unavailability of individual patient data that would allow correction for potential confounding factors such as age, gender, or additional genetic aberrations. Finally, different detection methods used in the studies included in the analysis may have different quality control issues.
In conclusion, our meta-analysis provides evidence that EGFR gene copy number is a prognostic marker for survival among patients receiving anti-EGFR mAbs for advanced colorectal cancer. Furthermore, according to our results, the prognostic ability of EGFR gene copy number appears to be significantly stronger among those populations that received second-line or higher treatment.