| Colorectal cancer (CRC) is one of the most common malignant tumor. Recently, with the improvement of living condition and people's life-span, the incidence rate of CRC has been ascensused year by year. In developed countries and the developed district of our country, CRC has become the second malignant tumor. Yearly, more than one million of CRC cases airse in the world, about 500,000 people died of the disease. CRC is one of the most reasons of people died of malignant tumor.As the etiology of CRC in-depth study, people found that the incidence of CRC is a multi-phase, milti-factor cumulative effects. Gene mutations and signaling transduction pathway activation are significantly associated with development of CRC. Some data reported that signaling pathways downstream of epidermal growth factor receptor (EGFR) was one of the major risk factor of metastatic colorectal cancer (MCRC). EGFR is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers two main signaling pathways. These include the RAS/RAF/MAPK axis, which is mainly involved in cell proliferation, and the PI3K/AKt pathway, which is mainly involved in cell survival and motility. Overexpression of EGFR is found in a range of solid tumor types. Over the past decade, EGFR inhabitors—monoclonal antibodies targeting the extracellular domain have expanded the range of therapeutics options for CRC. Cetuximab (Erbitux, C225), the first anti-EGFR monoclonal antibody to be approved for clinical use for MCRC in 2004, is effective in combination with chemotherapy in the first-line or second-line treatment of MCRC. Previous study showed that response to cetuximab is confined to MCRC patients with wild-type KRAS. KRAS gene mutation is a predictive bio-marker of response to cetuximab in CRC. In clinical practice, there are still some patients with wild-type KRAS who do not response to cetuximab. So, there are some biological factors that can affect the efficiency of anti-EGFR antibodies. Some data have indicated that KRAS mutations are found earlier in tumor progression. It is well known that gene mutations and signaling pathway activation resulted in the development of CRC. Little is known the relationship between these gene mutations and clinical pathological parameters (age, gender, tumor location, Dukes'staging, TNM, histological and metastatic). Recently, it was showen that mutations in other downstream components of the EGFR signaling transduction pathway might affect the efficiency of response to cetuximab, but whether these potential predictive biomarkers are validated or not still needs further investigation. Therefore, predictive and prognostic factors are required urgently and would help physicians to predict the risk of metastasis and to enact the personalized treatment for CRC patients.Two hundred paraffin-embedded tumor specimens were collected from colorectal cancer patients who underwent resection of primary tumors at Xijing Hospital of Digestive Diseases from 2008 to 2009. The clinicopathological parameters of the 200 cases of the colorectal cancers were collected from surgical and pathological records. Genomic DNA was extracted from paraffin-embedded tumor specimens. Pyrosequencing assay was used to detect KRAS, BRAF and PIK3CA mutations. The associations of KRAS, BRAF and PIK3CA mutations in colorectal cancer with clinical pathologicoparameters were explored using theχ2 test and Fisher's exact test. In the present study, we aimed to investigate the molecular occurrence of KRAS, BRAF and PIK3CA mutations in the colorectal cancer patients and to study the association of these events with clinicopathological parameters. Notably, we also tried to explore the association of KRAS, PIK3CA bi-mutations with MCRC. We expected to find some new biomarkers which would help to predict the patient progression and prognosis of CRC, thus to give proper recommendations for the clinical therapeutics. In addition to, clinicopathological parameters and paraffin-embedded tumor speci-mens were collected from colorectal cancer patients treated with cetuximab at Xijing Hospital of Digestive Diseases. We detect KRAS, BRAF, PIK3CA mutations and PTEN expressions in CRC, and we evaluate the efficacy of every patient treated with cetuximab. Based on our study, we expect to find whether these gene mutations can accurately predict the response to cetuximab or not, which will provide the necessary adjustment for optimized personalized treat-ment.In our study, we found that Pyrosequecing, which has been shown to be more sensitive than regular sanger sequencing, is a relatively straightforward method for identification of gene mutations in paraffin-embedded tumor samples. We detected KRAS mutation in 63/200 patients (31.5%), BRAF mutation in 14/200 patients (7%) and PIK3CA mutation in 25/200 (12.5%) patients. KRASmutations were found mostly in codon 12 (26%, 52/200), while codon 13 mutations (5%, 10/200) were fewer than codon 12 mutations. We found a codon12.13 (0.5%, 1/200) bi-mutation, which has not been reported before. PIK3CA mutation occurred in exon 9 (6%, 12/200) and exon 20 (6.5%, 13/200). Frequencies of mutations in our samples are compatible with data from previous studies. Hence, we deduced that there are no significant differences between the mutation status of KRAS, BRAF and PIK3CA from the West and east. Using 200 colorectal cancer samples, we confirmed that both KRASand BRAF mutations are exclusive, but KRAS and mutations are coexistent. Our study indicated that KRAS, BRAF and PIK3CA mutations were not significantly correlated with age, gender, tumor location and pathological categories. However, our data revealed significant correlation between KRAS, PIK3CA mutations and Dukes' staging. It was found that KRAS and PIK3CA mutations were significantly associated with Dukes' staging. In contrast, no significant relationship was found between BRAF mutation and Dukes' staging. The higher the colorectal cancer staging, the more frequencies of KRAS or PIK3CA mutations occurred. Our data showed that PIK3CA mutation is associated with KRAS mutation. PIK3CA mutation mostly occurred in Dukes' D staging of KRAS-mutated tumors. These data suggested that the colorectal cancer with KRAS and PIK3CA bi-mutations are more likely to develop into liver metastasis.In our study, we collected patients treated with cetuximab and assessmented the efficacy according to RECIST code. The primary end–point of the study was objective response (OR). We collected paraffin-embedded tumor specimes and clinicopathological parameters and checked mutations of KRAS, BRAF and PIK3CA. The PTEN expression status was detected through immunohistochemistry. Our study indicated that PTEN expression is most located in cell nucleus, PTEN expression was significantly associated with the response to cetuximab. The objective response rate of patients with wild-type BRAF and PIK3CA is 62.5% (CR+PR). In contrast, the objective response rate of patients with mutation-type BRAF and PIK3CA are 0, 28.6%. So, we deduct that patients with BRAF, PIK3CAmutations and PTEN expression null have also been associated with resistance to cetuximab. BRAF, PIK3CA and PTEN could be as predictive bio-markers of response to cetuximab.Taken together, our findings indicated that KRAS and PIK3CA mutations were significantly associated with Dukes' staging. PIK3CA mutations were found mostly in Dukes' D staging of KRAS-mutated tumors. CRC patients with KRAS and PIK3CA bi-mutations are more likely to develop into liver metastasis. Hence, CRC patients with KRAS, PIK3CA mutation or bi-mutations might have a poor prognosis and KRAS and PIK3CA bi-mutations may become potential predictive and prognostic factors. In addition to, our data showed that BRAF, PIK3CA mutations and PTEN expression null have also been associated with resistance to cetuximab. These innovations not only help us realized the biological meaning of gene mutations in CRC, but also explore whether these gene mutations can accurately predict the response to cetuximab or not, which will provide the necessary adjustment for optimized personalized treatment. |
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