| Colorectal cancer (CRC) is the third most common cancer in the world and hepatic metastases is one of the main reasons of death in CRC patients. Multiple genetic alterations play an important role in the development of colorectal cancer, including somatic mutations in RAS-RAF/PI3K pathway and chromosome 20q amplification. The presence of mutations in RAS-RAF/PI3K pathway is predictive for a lack of response to treatments with monoclonal antibodies against the epidermal growth factor receptor (EGFR) in patients, and is closely related to patients’ clinical and pathological features, metastasis and survival. In addition, patients who carried 20q amplification would be likely to have the risk of liver metastasis compared with other patients.Most studies were presented in Western populations, but there were few researches involved the frequency of common mutations in KRAS exon 2, BRAF and PIK3CA in Chinese patients.Only one study reported the mutations in KRAS exon 3 and exon 4, NRAS exon 2 and exon 3, as these mutations displayed low frequency in previous literature. First, this study aimed to detect mutation frequencies and distributions of KRAS exon 2, exon 3 and exon 4, NRAS exon 2 and exon 3, BRAF and PIK3CA in Chinese CRC patients, and to assess their correlations with the clinicopathologic features and survival status. Second, we designed 3 BAC-DNA probes located in 20q arm and combined iFISH technology to analyze amplification of 20q in the primary tumor.353 consecutive patients diagnosed with colorectal cancer at Zhongda Hospital (Nanjing, China, N=214) and Jiangyin People’s Hospital (Jiangyin, China, N=139) affiliated to Southeast University (Nanjing, China) from 2007 to 2012, were included in our study. All patients had complete clinicopathologic data and follow-up data. Genomic DNA was extracted and mutations were detected by PCR-direct sequencing. Then we assessed the relationship between these mutations and clinicopathologic characteristics, metastasis and survival status. The main results are as follows:1. KRAS, NRAS, BRAF and P1K3CA mutations were identified in 52.7%,3.4%,4.5% and 13.1% CRC patients, respectively. Patients who were not having KRAS mutations in exon 2 but in other RAS mutations reach up to 14%.2. NRAS mutations occurred more frequently in female patients than in male patients(75% vs 25%,p=0.019). BRAF mutations revealed strong associations with worse differentiation grade (p=0.003), advanced TNM stage (p=0.001), deeper tumor invasive depth (p=0.019), higher lymph node metastasis(p=0.004), preoperative and postoperative distant metastasis (p=0.002; p=0.016). PIK3CA mutated tumors were more likely to be on colon(p=0.014).3. Mutations in KRAS exon 2 occurred more frequently in older patients (p=0.036) and were associated with lymph node metastasis (p=0.046). While KRAS exon 3 mutations were more likely to appear on lower TNM stage (p=0.011) and shallow tumor invasive depth (p=0.001) CRCs.4. BRAF mutations revealed strong associations with decreased overall survival (p<0.001). Compared with the PIK3CA or NRAS wild type patients in stage Ⅰ-Ⅲ, those with PIK3CA or NRAS mutations revealed higher distant metastasis rates (p=0.033; p=0.017). In multivariate analysis, PIK3CA and NRAS mutations remained as an independent prognostic marker for distant metastasis in stage Ⅰ-Ⅲ patients (PIK3CA-. HR=3.129,p=0.003; NRAS:HR=5.152, p=0.003).Our previous study had already identified the 20q amplification status in 40 patients without metastasis and 6 with hepatic metastases. In this study we collected 4 hepatic metastasis patients and 11 extra-hepatic metastasis patients. We designed 3 BAC-DNA probes along 20q arm and used iFISH technology to analyze 20q amplification. ROC curves were drawn and threshold value was confirmed to assess the relationship between 20q amplification and clinicopathological features, survival status and hepatic metastasis.The main results are as follows:We observed obvious difference among the experimental probe signal and positive controls in tumor glands of CRC patients. The average value of the 3 probes we designed on 20q was higher than positive controls, suggesting that the amplified region was across from 20q12 to 20qter. Probe 20q13.2/20p12 is most suited to evaluate the 20q ampIification.But we have not yet found significant correlations between the status of 20q amplification and clinicopathological features, prognosis or hepatic metastasis.Very recently, China has published the first batch of high-throughput gene sequencing technology trials for tumor diagnosis and treatment, aiming at more precisely tumor treatment and survival prediction at gene level. We should actively look for more and more accurate molecular biomarkers for better the choices of targeted drugs, and better assessment of metastasis and prognosis. Our data provided important informations for predicting the effectiveness of anti-EGFR targeted drugs, especially the acculumative mutation frequency in RAS-RAF/PI3K pathway reached to 62.3%, which suggested that more than half of patients may not benefit from targeted therapy. Patients who took targeted drugs without genetic tests would suffer severe adverse reaction and bear the expensive cost of drugs if they had mutations. |
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