KRAS, NRAS, BRAF 和 PIK3CA基因突变在1100名中国结直肠癌中的分析

KRAS，NRAS，BRAF和PIK3CA基因突变已成为评价结直肠癌的重要组成部分。这项研究的目的是筛选中国结直肠癌患者（CRC）的这些基因突变，并探讨其突变与临床病理参数的相关性。我们在KRAS（外显子2，3和4）NRAS（外显子2，3和4）PIK3CA（外显子20）和BRAF（外显子15）基因测试突变，使用逆转录酶 - 聚合酶链反应（RT-PCR）和Sanger测序分析1110名中国结直肠癌患者，这些患者已在中国不同地区的医院的接受过一次手术切除。

患者的KRAS，NRAS，BRAF和PIK3CA基因突变分别为45.4％，3.9％，3.1％和3.5％。KRAS突变与粘液性亚型和更多的分化有关，而BRAF突变与右侧肿瘤和较差的分化有关。我们的研究结果揭示了KRAS，NRAS，PIK3CA和BRAF在中国CRC患者和西方国家患者之间的突变热点的遗传图谱的差异，尽管亚洲其他国家的一些病人的基因是共用。

简介：

结直肠癌是全球第三大最常见的恶性肿瘤。几年来中国结直肠癌的发病率和死亡率均上升。2010年，中国的结直肠癌的粗测发病率为20.90/10万人，粗统计死亡率为10.1/10万人，在所有癌症中排名第六。虽然手术仍是治疗局部肿瘤的有效治疗方法，化疗药物组合用于延长晚期病人的整体和无病生存。

结直肠癌发展得原因是多个基因突变、多个步骤积累造成的。 多个信号通路激活，特别是RAS-RAF-MAPK和PI3K-PTEN-AKT信号通路，在调节细胞增殖、血管生成、细胞运动和细胞凋亡中起到重要的作用。 因此，在肿瘤抑制基因和原癌基因如KRAS，NRAS，BRAF及PIK3CA的突变积累，显著有助于结直肠癌CRC的发展。

在转移性结直肠癌的治疗中，表皮生长因子受体的单克隆抗体（EGFR），例如西妥昔单抗和帕尼单抗，已自2004年以来用于临床实践。 大约30％至45％的CRC肿瘤有KRAS突变，KRAS突变与EGFR 抗体耐药性有关。而具有野生型KRAS基因似乎是对治疗有反应的先决条件，这并不一定能预测抗EGFR单克隆抗体是否有响应，表明额外的遗传改变可能会加大患者对药物的非响应性。除了KRAS基因突变外，EGFR突变的下游通路的效应器突变，例如BRAF, NRAS，包括 PI3K 信号通路的组成部分， 可能减弱anti-EGFR 抗体的效果。

迄今为止，众多的调查EGFR-RAS-RAF途径和PI3K途径的突变，并揭示出这些基因突变的不同分布格局。但是，与之前的报道在不一致的是这些研究需要一个更大的样本，中国的多中心的研究。

在本研究中，我们的目的是使用逆转录聚合酶链反应（RT-PCR）和Sanger测序中国1110名CRC患者的基因突变，并确定这些突变的频率，以评估KRAS，NRAS，BRAF和PIK3CA突变，及这些突变与临床病理参数之间的关系。

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

文章来源：http://www.nature.com/articles/srep18678

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

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.

Introduction

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 sites 1. 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 antibodies 7. 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 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 antibodies 9,10,11,12,13,14.

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.

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

文章来源：http://www.nature.com/articles/srep18678

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