Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer
Concomitant mutations and splice variants in KRAS and BRAF demonstrate complex perturbation of the Ras/Raf signalling pathway in advanced colorectal cancer
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DOI：
10.1136/gut.2008.159137
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发表时间：
2009-09-01
期刊：
影响因子：
24.5
通讯作者：
Ilyas, M.
中科院分区：
文献类型：
--
作者：
Seth, R.;Crook, S.;Ilyas, M.
背景和目标：KRAS和BRAF突变发生在结直肠癌（CRC）中，被认为是激活RAS/RAF/MEK/ERK通路的相互排斥的方法。该途径是治疗靶点，KRAS突变可以预测肿瘤的反应性。本研究旨在探讨24例结直肠癌细胞系和29例晚期结直肠癌中KRAS和BRAF突变的关系。通过逆转录-PCR（RT-PCR）和测序证实突变的表达。结果：79%的细胞株和59%的CRC中存在KRAS或BRAF突变。在细胞系中，54%的病例发生KRAS突变（密码子12/13为62%，其他密码子为38%）。4个细胞系有纯合突变。在29%的细胞系中仅检测到杂合BRAF突变。V600 E突变最常见，与CIMP+状态相关（p=0.005）。还发现了密码子529和581处的突变，并且在一个病例中，BRAF和KRAS突变同时发生。出乎意料的是，在5/24（21%）的细胞系中发现了BRAF剪接变体（具有预测的激酶死亡蛋白）。在晚期CRC中，48%的病例发生KRAS突变（64%密码子12/13，36%其他密码子），10%发生BRAF突变（66% V600 E，33%外显子11）。一个复合KRAS/BRAF突变是没有看到。结论：中断Ras/Raf信号是常见的结直肠癌。纯合子KRAS突变和伴随的KRAS/BRAF突变可能指示基因剂量效应。BRAF剪接变异体的意义尚不确定，但可能代表另一层复杂性。最后，如果KRAS突变用于预测性测试，则可能需要筛选整个基因，因为突变发生在密码子12/13之外。
Background and aims: KRAS and BRAF mutations occur in colorectal cancers (CRCs) and are considered mutually exclusive methods of activating the RAS/RAF/MEK/ERK pathway. This pathway is a therapeutic target and KRAS mutation may predict tumour responsiveness. The purpose of this study was to investigate the relationship between KRAS and BRAF mutations in 24 CRC cell lines and 29 advanced CRCs.Methods: KRAS and BRAF mutations were detected using high resolution melting and sequencing. Expression of mutations was confirmed by reverse transcription-PCR (RT-PCR) and sequencing. CpG island methylator phenotype (CIMP) was tested by methylation-specific PCR.Results: KRAS or BRAF mutation occurred in 79% of cell lines and 59% of CRCs. In the cell lines, KRAS mutations occurred in 54% of cases (with 62% in codons 12/13 and 38% in other codons). Four cell lines had a homozygous mutation. Only heterozygous BRAF mutations were detected in 29% cell lines. The V600E mutation occurred most commonly and was associated with CIMP+ status (p=0.005). Mutations at codons 529 and 581 were also found and, in one case, BRAF and KRAS mutation co-occurred. Unexpectedly, BRAF splice variants (with a predicted kinase-dead protein) were found in 5/24 (21%) cell lines. In advanced CRCs, KRAS mutations occurred in 48% of cases (64% codons 12/13, 36% other codons) and BRAF mutations in 10% (66% V600E, 33% exon 11). A compound KRAS/BRAF mutation was not seen.Conclusions: Disrupted Ras/Raf signalling is common in CRC. Homozygous KRAS mutations and concomitant KRAS/BRAF mutations may be indicative of a gene dosage effect. The significance of BRAF splice variants is uncertain but may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene may need to be screened as mutations occur outside codons 12/13.