Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases

www.nature.com/scientificreports OPEN received: 11 August 2015 accepted: 23 November 2015 Published: 22 December 2015 Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases Jing Zhang1,*, Jianming Zheng2,*, Yinghong Yang3,*, Junliang Lu1, Jie Gao1, Tao Lu1, Jian Sun1, Hui Jiang2, Yan Zhu2, Yuhui Zheng3, Zhiyong Liang1 & Tonghua Liu1 Mutations in genes such as KRAS, NRAS, BRAF and PIK3CA have become an important part of colorectal carcinoma evaluation. The aim of this study was to screen for mutations in these genes in Chinese patients with colorectal cancer (CRC) and to explore their correlations with certain clinicopathological parameters. We tested mutations in the KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4), PIK3CA (exon 20) and BRAF (exon 15) genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing in a large cohort of 1,110 Chinese CRC patients who underwent surgical resection at one of three major teaching hospitals located in different regions of China. The prevalence rates of KRAS, NRAS, BRAF and PIK3CA mutations were 45.4%, 3.9%, 3.1% and 3.5%, respectively. Mutant KRAS was associated with the mucinous subtype and greater differentiation, while mutant BRAF was associated with right-sided tumors and poorer differentiation. Our results revealed differences in the genetic profiles of KRAS, NRAS, PIK3CA and BRAF at mutation hotspots between Chinese CRC patients and those of Western countries, while some of these gene features were shared among patients from other Asian countries. Colorectal cancer (CRC) is the third most common malignancy worldwide. In recent years, the morbidity and mortality due to CRC have risen in the Chinese population. In 2010, the crude incidence rate of CRC in China was 20.90/100,000, and the crude mortality rate was 10.1/100,000, ranking 6th among all cancer sites1. Although surgery remains the only curative method for patients with localized tumors, several combinations of chemotherapeutic drugs are used to extend overall and disease-free survival for those with advanced disease2. The development of CRC is a multistep process that results from the accumulation of several genetic alterations. The activation of multiple signaling pathways, specifically RAS-RAF-MAPK and PI3K-PTEN-AKT, plays an important role in regulating cell proliferation, angiogenesis, cell motility, and apoptosis3,4. Accordingly, the accumulation of mutations in tumor suppressor genes and proto-oncogenes participating in these signaling pathways, such as KRAS, NRAS, BRAF and PIK3CA, significantly contributes to the development of CRC5,6. In the treatment of metastatic colorectal cancer, monoclonal antibodies against epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, have been used in clinical practice since 2004. Approximately 30% to 45% of CRC tumors harbor a KRAS mutation, and mutant KRAS is associated with resistance to anti-EGFR antibodies7. While wild-type KRAS appears to be a prerequisite for the response to treatment, it does not necessarily predict the response to anti-EGFR monoclonal antibodies7,8, indicating that additional genetic alterations might contribute to this non-responsiveness. In addition to KRAS mutations, mutations in other downstream effectors 1 Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, People’s Republic of China. 2Department of Pathology, Changhai Hospital of Shanghai, Shanghai, People’s Republic of China. 3Department of Pathology, Fujian Medical University Union Hospital, Fuzhou, People’s Republic of China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Z.L. (email: ) or T.L. (email: ) Scientific Reports | 5:18678 | DOI: 10.1038/srep18678 1 www.nature.com/scientificreports/ of the EGFR signaling pathway, such as BRAF, NRAS, and components of the PI3K signaling pathway, potentially exert negative effects on the response to anti-EGFR antibodies9–14. To date, numerous investigations into the mutational status of components in the EGFR-RAS-RAF pathway and the PI3K pathway have been conducted and have revealed a diverse distributional pattern of mutations in these genes. However, inconsistency in the prevalence of certain mutations reported in these studies elicits the need for a multicenter study in China in an even larger sample. In the present study, we aimed to evaluate KRAS, NRAS, BRAF and PIK3CA mutations using both reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing in 1,110 samples from Chinese patients with CRC and to determine the frequencies of these mutations and the relationships between these mutations and clinicopathological parameters. Materials and Methods Samples. The records of all patients diagnosed with CRC from January 2012 to December 2014 at the Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, (Beijing, China; 514 cases), the Department of Pathology, Changhai Hospital of Shanghai, Second Military Medical University (Shanghai, China; 299 cases), and the Department of Pathology, Fujian Medical University Union Hospital (Fujian, China; 297 cases) were retrieved. The following exclusion criteria were applied: patients who underwent neoadjuvant therapy before surgery, unavailability of paraffin block specimens for pathology and insufficient clinical information. A total of 1,110 formalin-fixed paraffin-embedded (FFPE) CRC tissue samples were evaluated. Tumor sections from FFPE tissue samples were stained with hematoxylin–eosin (H&E) and reviewed by two experienced histopathologists independently. Clinicopathological information was obtained by reviewing the medical records in detail and noting the age (≤ 60 or > 60 years), sex (male or female), tumor site (right, left colon or rectum), histological type, differentiation, depth of invasion, lymph node metastasis, distant metastasis and TNM stage. This study was conducted with the approval of the Ethics Committee of all three hospitals, and informed consent was obtained from all patients. The methods were carried out in accordance with the approved guidelines. DNA extraction. Sections (5 μ m thick) were cut from paraffin-embedded tumor tissue blocks and stained with H&E for histopathological examination. Each sample was evaluated by two experienced pathologists. To obtain maximal tumor DNA, we chose tumor-rich paraffin block specimens whose tumor components were greater than 70% and the amount of stroma was less than 30%. For DNA isolation, 5-μ m-thick sections were used for each case. The H&E section was used as a reference, and tumor-rich regions of the sections were trimmed off from the slides based on their respective H&E staining patterns and transferred to lysate buffer (included in the DNA purification kit). DNA in the colle (...truncated)