Gene Mutations In EGFR Signaling Network And Their Association With Survival In Patients With Metastatic Colorectal Cancer

BackgroundThe protein encoded by epidermal growth factor receptor (EGFR) gene is a transmembrane glycoprotein, which is a member of the protein kinase superfamily. This protein is a receptor for the epidermal growth factor (EGF) family. EGFR is a protein that binds to EGF, activating the intracellular downstream signaling pathways, of which the KRAS/RAF/MEK/ERK pathway and the PI3K/AKT pathway are very important. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation, migration, survival and angiogenesis through the two pathways mentioned above.EGFR signaling pathway is an important target for molecular targeted therapy due to the fact that it play an important role in the occurrence and development of malignant tumors. The study of biological molecules and drugs development for EGFR and its downstream signal transduction pathway have become hot spot. Anti-EGFR monoclonal antibodies (MAbs) such as cetuximab and panitumumab, which are the most representative drugs of molecular targeted therapy in metastatic colorectal cancer (mCRC), have been approved for clinical application. Studies showed that KRAS gene played an important role in inhibiting apoptosis and accelerated tumor cell proliferation. KRAS gene status was an effective indicator to predict the efficacy of anti-EGFR therapy. Related research suggested that KRAS gene mutation could lead to failure of anti-EGFR monoclonal antibody therapy. It has proved in several II and III phase clinical trials that only the patients bearing KRAS wild-type gene could benefit from anti-EGFR monoclonal antibody treatment. Therefore, the experts of American Society of Clinical Oncology suggested that all the patients with metastatic colorectal cancer preparing to accept anti-EGFR monoclonal antibody treatment should receive the test of KRAS gene status. The results brought about the consideration that EGFR-targeted therapy should be restricted to patients bearing wild-type KRAS. However, selecting patients for the EGFR-targeted therapy by KRAS status of tumor was not enough. Because some of the patients bearing wild-type KRAS tumors failed to respond to the EGFR-targeted therapy, researcher suggested that there might be other molecular determinants to be identified. Therefore, how to predict the efficacy of anti-EGFR treatment and to choose the suitable patients through the clinical features, pathological features and gene mutation status have attracted widespread attention and become urgent problems for the clinical use of EGFR monoclonal antibody.Previous studies have shown that BRAF, PIK3CA were the targets for the genetic mutations of colorectal cancer and related with EGFR targeted therapy. Therefore, research on the various genes mutation frequency of the EGFR signal transduction pathway in colorectal cancer will help to predict the efficacy of EGFR targeted therapy. That is to say, determination the prevalence of mutations in EGFR signaling network in mCRC will be useful to predict the response to EGFR targeted therapy. Although a number of clinical investigations from western countries have reported the prevalence of mutations in KRAS and BRAF in patients with CRC, there were seldom studies in EGFR and PIK3CA. Research data were not sufficient for the relationship between the EGFR pathway gene mutation status and the overall survival of patients who did not receive EGFR targeted therapy, and the existing research results were different. Most of these studies aimed at a single gene mutation status and could not systematically and comprehensively explain the relationship between the gene mutations of EGFR signal transduction pathway and the overall survival. In addition, the studies in Chinese patients remain unknown. As the gene status exist significant differences in different races, this difference could lead to significant different result for targeted therapy. One of the global multi-center lung cancer targeted therapy clinical studies has confirmed the hypothesis. Therefore, it is necessary to study gene status of EGFR signal transduction pathway and their impact on overall survival.Due to the heterogeneity of tumors, there may be genotype inconsistencies between the primary and metastatic tumors. It has long been concerned that tumor heterogeneity led to inconsistencies in phenotype. The study of Italiano showed that EGFR protein expression and gene status of non-small cell lung cancer between primary and metastatic tumors were not uniform by immunohistochemical staining and FISH method. Kalikaki also found the gene status of EGFR and KRAS were not consistent in non-small cell lung cancer. The study of Oliveira showed that the frequency of KRAS mutation was significant higher in lymph node metastases than in primary carcinomas in patients with CRC. These studies showed that deducing the gene status in metastatic tumor only based on primary tumor was not accurate. Because the KRAS gene status affected the efficacy of EGFR monoclonal antibody therapy in patients with mCRC, it is very important to confirm the consistency of KRAS gene mutations between primary and metastatic tumors in colorectal cancer.There are a variety of detecting methods for KRAS gene mutation. These methods include single strand conformation polymorphism analysis, mutant-enriched PCR coupled sequencing, pyrosequencing, dideoxy sequencing. Quantitative-PCR and PCR dot-blot hybridization. Studies have shown that the different sensitivity of method may cause differences in test results. The study of Whitehall showed that only 62.2%(46/74) of the results were consistent in the all six kinds of methods. The lowest frequency of KRAS mutations was 32.9% and the highest was 59.5%. In addition, DNA quality and relative content of tumor cells in samples are also important determinant factors, which should be paid more attention to during the work of gene mutations detecting.Our study were as follows:(1)We assess mutations of EGFR, KRAS, BRAF and PIK3CA in patients with mCRC simultaneously, evaluate these molecular markers individually and in combination for their ability to predict the prognosis of overall survival in patients, which may improve the study of individual therapy in Chinese mCRC patients. (2) In order to study the difference of KRAS gene mutation status between primary and metastatic tumors, we conducted a study on KRAS gene mutation status in primary and corresponding metastatic tumors to understand the heterogeneity of tumors, which may impact on EGFR targeted therapy. (3) A preliminary comparative study about two kinds of KRAS gene mutation detecting methods were conducted to understand their effect on testing result. Through these studies, we hope to find some useful resource to improve the study of individual therapy in Chinese mCRC patients.Methods1. Frequencies of EGFR, KRAS, BRAF and PIK3CA MutationsTumor specimens were collected from 61 histologically confirmed mCRC patients with complete follow up data, who were selected from 2030 patients with colorectal cancer. DNA was extracted from paraffin-embedded tissue of colorectal cancer. We designed primers to amplify the right exons of EGFR, KRAS, BRAF and PIK3CA genes, which mutated most frequently, by PCR. Those PCR products were pyrosequenced and the frequencies of EGFR, KRAS, BRAF and PIK3CA Mutations were analyzed.2. The prognostic value of pathological parameters and gene mutation on overall survival of patientsKaplan-Meier and Cox proportional hazard models were used to assess the prognostic significance of mutations of KRAS, BRAF and PIK3CA genes for survival and adjusted for age, gender, tumor location, metastatic lesions, and treatment regimens.3. The research of KRAS gene status of Primary and liver metastatic tumors of colorectal cancer46 samples of primary and liver metastatic tumors were collected, which obtained from biopsy and surgery. The samples were marked and tumor slices from each specimen were obtained, then microdissected tumor tissue and extracted DNA. The primers of codon 12 and 13 mutation status of KRAS gene were designed and gene mutaiton status were detected by mutant-enriched PCR coupled sequencing.4. A comparative study of two methods for detection of KRAS gene mutations13 cases of tissue samples were selected. The KRAS gene mutation status was detected by mutant-enrichment PCR coupled sequencing and pyrosequencing assay simultaneously.Results1. Frequencies of EGFR, KRAS, BRAF and PIK3CA MutationsIn the samples collected from 61 mCRC patients, we detected mutations of EGFR, KRAS, BRAF and PIK3CA in 0 (0%),12 (19.7%),3(4.9%) and 3 (4.9%) patients, respectively. Only one case bears KRAS and PIK3CA mutations at the same time.2. The prognostic value of pathological parameters and gene mutation on overall survival of patientsIn a univariate Cox regression analysis, we found that only age, the number of metastatic lesions and treatment regimens were significant prognostic factors for survival time. Patients younger than 65 years indicate a poor prognosis (HR 2.083, P=0.042). In contrast to patients with 3 or more than 3 metastatic lesions, patients with only one metastatic lesion had a lower HR of death (HR 2.083, P=0.042), and similarly, patients received multidisciplinary therapy had a lower HR of death (HR 2.083, P=0.042). No any significant associations were noted between the mutations of KRAS, BRAF and PIK3CA and the HR of death of the patients.Results of Kaplan-Meier survival function analysis grouped by treatment regimens, or mutation of at least 1 of 3 genes, or metastatic location, or metastatic lesions showed that there were no significant differences in cumulated survival between patients with mutations in any of the 3 genes and those with wild-type ones. In a multivariate analysis using Cox model, we noted that only treatment regimens (P =0.001) and age (P=0.017) were independent factors to predict HR for death.3. The research of KRAS gene status of primary and liver metastatic tumors of colorectal cancerKRAS gene mutations were founded in 18 (39.1%) cases of the primary tumors and 19 cases (41.3%) of metastatic tumors. In the 18 cases with mutated KRAS gene in the primary tumor tissues, only one case of KRAS gene was wild-type in its corresponding hepatic metastasis. In the 28 cases with wild-type KRAS gene in the primary tumor tissues, only two cases of KRAS gene mutated in its corresponding liver metastasis. Statistical analysis showed that there was no significant difference in the status of KRAS gene between the primary tumor and liver metastasis.4. A Comparative study of two methods for detection of KRAS gene mutationWe compare mutant-enrichment PCR coupled sequencing and pyrosequencing assay. In 13 cases of samples, mutated KRAS was found in 8 cases by mutant-enriched PCR coupled sequencing, whereas only 6 samples by pyrosequencing assay.Conclusions1. The frequency of EGFR mutation was low, and consistented with the foreign reports. However, the results by pyrosequencing assay showed that the mutation frequencies of KRAS, BRAF and PIK3CA gene were lower compared with the results of the foreign reports. The results suggest that the Chinese population may be more suitable for the EGFR monoclonal antibody targeted therapy.2. There was no significant correlation between the KRAS, BRAF and PIK3CA gene mutations status in EGFR signal transduction network and overall survival. Even if removing the impact of other factors by the application of multivariate analysis methods, we still got the same results. Patients with less metastatic lesions, or older than 65 years, would have a lower HR of death. Multidisciplinary treatment can effectively improve the prognosis of patients.3. Although KRAS gene mutations between primary and liver metastatic tumors were not entirely consistent, there was no statistically significant difference. These results confirmed the consistency of KRAS gene mutations between primary and liver metastatic tumors in colorectal cancer. It will be helpful for the clinical application of anti-EGFR monoclonal antibody. In order to obtain more convincing results, large sample research should be carried out. 4. There was no difference between mutant-enriched PCR coupled sequencing and pyrosequencing assay in detection of KRAS gene mutations. But pyrosequencing assay failed to detect two cases of KRAS mutation, which were found to bear mutations by using mutant-enriched PCR coupled sequencing. The results may be related with different sample preparation and DNA extraction methods.